o  m 


Issued  March  24.  1013< 

U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY.— BULLETIN  161. 

A.  D.  MELVIN,  CHIEF  OF  BUREAU. 


STUDY  OF  THE  BACTERIA  WHICH 
SURVIVE  PASTEURIZATION. 


BY 

S.  HENRY  AYERS, 
Bacteriologist^ 

AND 

WILLIAM  T.  JOHNSON,  JR., 

Scientific  Assistant,  Dairy  Division. 


lifornia 

ional 

ility 


WASHINGTON: 

GOVERNMENT    PRINTING   OFFICE. 
1913. 


Issued  March  24,  1913. 

U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY.— BULLETIN  161. 
A.  D.  MELVIN,  CHIEF  OF  BUREAU. 


A  STUDY  OF  THE  BACTERIA  WHICH 
SURVIVE  PASTEURIZATION. 


BY 

S.  HENRY  AYERS, 

Bacteriologist, 

AND 

WILLIAM  T.  JOHNSON,  JR. 

Scientific  Assistant,  Dairy  Division. 


WASHINGTON: 

GOVERNMENT   PRINTING   OFFICE. 
1913. 


THE   BUREAU    OF   ANIMAL   INDUSTRY. 


Chief:  A.  D.  MELVIN. 

Assistant  Chief:  A.  M.  FARRINGTON. 

Chief  Clerk:  CHARLES  C.  CARROLL. 

Animal  Husbandry  Division:  GEORGE  M.  ROMMEL,  chief. 

Biochemic  Division:  M.  DORSET,  chief. 

Dairy  Division:  B.  H.  RAWL,  chief. 

Field  Inspection  Division:  R.  A.  RAMSAY,  chief. 

Meat  Inspection  Diinsion:  R.  P.  STEDDOM,  chief. 

Pathological  Division:  JOHN  R.  MOHLER,  chief. 

Quarantine  Division:  RICHARD  W.  HICKMAN,  chief. 

Zoological  Division:  B.  H.  RANSOM,  chief. 

Experiment  Station:  E.  C.  SCHROEDER,  superintendent. 

Editor:  JAMES  M.  PICKENS. 

DAIRY  DIVISION. 

B.  H.  RAWL,  chief. 

HELMER  RABILD,  in  charge  of  Dairy  Farming  Investigations. 
S.  C.  THOMPSON,  TO  charge  of  Dairy  Manufacturing  Investigations. 
L.  A.  ROGERS,  in  charge  of  Research  Laboratories. 
ERNEST  KELLY,  in  charge  of  Market  Milk  Investigations. 
ROBERT  McADAM,  in  charge  of  Renovated  Butter  Inspection. 
2 


ADDITIONAL  COPIES  of  this  publication 
-tx  may  be  procured  from  the  SUTEKINTENIV 
KNT  or  DOCUMENTS,  Government  Printing 
Office,  Washington,  D.  C.,  at  10  cents  per  copy 


LETTER  OF  TRANSMITTAL. 


U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY, 

Washington,  D.  C.,  October  7,  1912. 

SIR:  I  have  the  honor  to  transmit  herewith  for  publication  in  the 
bulletin  series  of  this  bureau  a  manuscript  entitled  "A  Study  of  the 
Bacteria  Which  Survive  Pasteurization,"  by  Messrs.  S.  Henry  Ayers 
and  William  T.  Johnson,  jr.,  of  the  Dairy  Division. 
Respectfully, 

A.  D.  MELVIN, 

Chief  of  Bureau. 
Hon.  JAMES  WILSON, 

Secretary  of  Agriculture. 

3 


CONTENTS. 


Page. 

Introduction 9 

Objects  of  this  investigation 10 

Methods 11 

Temperatures  used  throughout  the  country 12 

Bacterial  reductions  by  laboratory  pasteurization,  using  the  ''holder  "  process . .  13 

Bacterial  reductions  at  60°  C.  (140°  F.)  and  65.6°  C.  (150°  F.) 13 

Bacterial  reductions  at  62.8°  C.  (145°  F.) 15 

The  control  of  pasteurization 16 

Effect  of  long  heating  on  bacteria  in  milk 17 

Three  hours'  pasteurization 17 

Six  hours'  pasteurization 18 

Effect  of  sudden  cooling  on  the  bacteria  in  pasteurized  milk 20 

Groups  of  bacteria  which  survive  pasteurization 21 

Grades  of  milk  studied 21 

Methods ....  21 

The  bacterial  groups  in  grade  A  milk  before  and  after  pasteurization  for 

30  minutes  at  62.8°  C.  (145°  F.) 24 

The  bacterial  groups  in  grade  A  milk  before  and  after  pasteurization  for 

30  minutes  at  7 1.1°  C.  (160°  F.) 25 

The  bacterial  groups  in  grade  B  milk  before  and  after  pasteurization  for 

30  minutes  at  62.8°  C.  (145°  F.) 25 

The  bacterial  groups  in  grade  C  milk  before  and  after  pasteurization  for 

30  minutes  at  62.8°  C.  (145°  F.) 26 

The  bacterial  groups  in  milk  pasteurized  at  high  temperatures 27 

The  activity  of  bacteria  of  the  various  groups  isolated  from  raw  and  pasteurized 
milk  measured  by  their  ability  to  produce  changes  in  litmus  milk  after  dif- 
ferent lengths  of  incubation 28 

Grade  A  milk  before  and  after  pasteurization  for  30  minutes  at  62.8°  C. 

(145°  F.) ,. 28 

Grade  A  milk  before  and  after  pasteurization  for  30  minutes  at  71.1°  C. 

(160°  F) 30 

Grade  B  milk  before  and  after  pasteurization  for  30  minutes  at  62.8°  C. 

(145°  F.) 31 

Grade  C  milk  before  and  after  pasteurization  for  30  minutes  at  62.8°  C. 

(145°  F.) 32 

Comparison  of  the  percentage  of  acid-forming  bacteria  in  raw  and  pasteurized 

milk 33 

Comparison  of  the  percentage  of  peptonizing  bacteria  in  raw  and  pasteurized 

milk 34 

Average  percentages  of  the  bacterial  groups  of  raw  milk  which  survive  pasteuri- 
zation   34 

5 


6  CONTENTS. 


The  effect  of  pasteurization  on  the  Dumber  of  bacteria  of  different  groups  in 

milk 35 

The  bacterial  development  in  milk  pasteurized  in  the  laboratory  and  held  at 

different  temperatures 37 

Qualitative  study  of  the  groups  of  bacteria  which  survive  pasteurization 43 

The  acid-forming  group  of  bacteria 43 

The  inert  group  of  bacteria 45 

The  alkali-forming  group  of  bacteria 45 

The  peptonizing  group  of  bacteria 47 

The  gas-forming  bacteria 47 

Thermal  death  points  of  bacteria  which  survive  pasteurization 51 

The  possible  use  in  pasteurization  of  acid-forming  bacteria  of  high  thermal 

death  point 52 

Qualitative  study  of  the  complete  bacterial  flora  of  one  sample  of  pasteurized 

milk 53 

Summary 58 

Conclusions. 61 

Appendix.    Tables 65 


ILLUSTRATIONS. 

Page. 
FIGURE  1.  Bacterial  reduction  during  pasteurization  for  three  hours  at  54.4° 

C.  (130°  F.),  57.2°  C.  (135°  F.),  and  60°  C.  (140°  F.) 17 

2.  Bacterial  reduction  during  pasteurization  for  six  hours  at  62.8°  C, 

(145°  F.).     Plotted  after  one-half,  three,  and  six  hours. . . ...         19 

3.  Bacterial  reduction  during  pasteurization  for  six  hours  at  62.8°  C. 

(145°  F.).     Plotted  every  half  hour 20 

4.  Comparison  of  the  milk-tube  and  plate  method  for  the  determination 

of  the  bacterial  groups  in  milk 23 

5.  Bacterial  groups  of  grade  A  milk  before  and  after  pasteurization  for 

30  minutes  at  62.8°  C.  (145°  F.) 24 

6.  Bacterial  groups  in  grade  A  milk  before  and  after  pasteurization  for 

30  minutes  at  71.1°  C.  (160°  F.) 25 

7.  Bacterial  groups  in  grade  B  milk  before  and  after  pasteurization 

for  30  minutes  at  62.8°  C.  (145°  F.) -    26 

8.  Bacterial  groups  in  grade  C  milk  before  and  after  pasteurization  for 

30  minutes  at  62.8°  C.  (145°  F.) 27 

9.  Bacterial  groups  which  survive  pasteurization  for  30  minutes  at  76.7° 

C.  (170°  F.),  82.2°  C.  (180°  F.),  87.8°  C.  (190°  F.),  and  93.3°  C 
(200°  F.) 28 

10.  Changes  in  the  bacterial  group  relations  in  grade  A  milk  when  de- 

termined by  litmus  milk  reactions  after  different  lengths  of  incu- 
bation.   Milk  pasteurized  at  62.8°  C.  (145°  F.)  for  30  minutes.  . .        29 

11.  Changes  in  the  bacterial  group  relations  in  grade  A  milk  when  de- 

termined by  litmus  milk  reactions  after  different  lengths  of  incu- 
bation.    Milk  pasteurized  at  71.1°  C.  (160°  F.)  for  30  minutes. . .         30 

12.  Changes  in  the  bacterial  group  relations  in  grade  B  milk  when  de- 

termined by  litmus  milk  reactions  after  different  lengths  of  incu- 
bation          31 

13.  Changes  in  the  bacterial  group  relations  in  grade  C  milk  when  de- 

termined by  litmus  milk  reactions  after  different  lengths  of  incu- 
bation          33 

14.  Comparison  of  the  percentage  of  the  acid-forming  bacteria  in  raw  and 

pasteurized  milk 33 

15.  Comparison  of  the  percentage  of  the  peptonizing  bacteria  in  raw  and 

pasteurized  milk 34 

16.  Daily  changes  in  the  bacterial  group  relations  in  pasteurized  milk. 

Sample  G  held  at  room  temperature " 39 

17.  Daily  changes  in  the  bacterial  group  relations  in  pasteurized  milk. 

Sample  G  held  in  the  ice  box 39 

18.  Daily  changes  in  the  bacterial  group  relations  in  pasteurized  milk. 

Sample  H  held  at  room  temperature 40 

7 


8  ILLUSTRATIONS. 

Page. 
FIGURE  19.  Daily  changes  in  the  bacterial  group  relations  in  pasteurized  milk. 

Sample  II  held  in  ice  box 41 

20.  Daily  changes  in  the  bacterial  group  relations  in  pasteurized  milk. 

Sample  K  held  at  room  temperature 42 

21.  Daily  changes  in  the  bacterial  group  relations  in  pasteurized  milk. 

Sample  K  held  in  the  ice  box 42 

22.  The  acid-forming  group  of  bacteria 44 

23.  The  alkali-forming  group  of  bacteria 45 

24.  The  peptonizing  group  of  bacteria 46 

25.  Apparatus  for  the  determination  of  gas  production  in  milk 48 

26.  Gas  produced  by  culture  Z 49 

27.  A  series.     Cultures  from. milk  pasteurized  at  60°  C.  (140°  F.)  for  30 

minutes 54 

28.  B  series.     Cultures  from  milk  pasteurized  at  65.6°  C.  (150°  F.)  for 

30  minutes 56 

29.  The  hypothetical  relation  of  the  bacterial  groups  to  raw  and  pas- 

teurized milk 62 

30.  The  hypothetical  relation  of  the  bacterial  groups  in  raw  and  pas- 

teurized milk. ...  63 


A  STUDY  OF  BACTERIA  WHICH  SURVIVE  PASTEURIZATION. 


INTRODUCTION. 

The  bacteria  which  survive  the  process  of  pasteurization  are  of  great 
importance,  since  they  form  the  foundation  for  the  subsequent  bac- 
terial development.  It  is  well  known  that  the  most  efficient  pasteu- 
rization will  not  destroy  all  of  the  microorganisms  in  milk.  The 
process  must,  then,  leave  a  certain  number  of  bacteria  which  it  is 
impossible  to  destroy  at  the  temperatures  employed. 

The  prevailing  view  has  been  that  the  organisms  left  after  heating 
were  largely  of  the  peptonizing  spore-forming  type,  with  some  few 
inert  forms.  This  idea  was  based  on  the  belief  that  all  vegetative 
cells  were  destroyed  at  temperatures  below  the  minimum  pasteurizing 
temperature  of  60°  C.  (140°  F.). 

Theoretically,  then,  only  spore-forming  organisms  should  survive 
pasteurization.  This  view  was  strengthened  by  numerous  investi- 
gations of  the  bacterial  flora  of  boiled  milk  or  milk  pasteurized  at 
extremely  high  temperatures.  As  lower  temperatures  came  gradu- 
ally into  use  investigations  indicated  that  vegetative  cells  might 
have  higher  thermal  death  points  than  was  generally  believed, 
Russell  and  Hastings  1  found  a  micrococcus  which  was  capable  of 
standing  a  temperature  of  76°  C.  (168.8°  F.)  for  10  minutes.  Other 
investigators  pointed  out  that  lactic  acid  bacteria  occasionally  ap- 
peared in  pasteurized  milk  and  yet  the  thermal  death  point  of  the 
lactic  acid  bacteria  was  stated  to  be  in  the  neighborhood  of  57.2°  to  60° 
C.  (135°-140°  F.).  Rogers3  found  that  in  milk  pasteurized  at  85° 
C.  (185°  F.)  by  the  "flash"  process  lactic  acid  bacteria  occasionally 
survived.  Marshall 3  states  "It  is  largely  supposed  that  in  pasteur- 
ized milk  the  lactic  acid  bacteria  are  killed.  This  is  largely  true  but 
not  universally."  Maz4*  found  that  the  ordinary  lactic  acid  bacteria 

i  Russell,  IT.  L.,  and  Hastings,  E.  G.  A  micrococcus,  the  thermal  death  limit  of  which  is  76°  C.  Cen- 
tralblatt  fiir  BrJrtorioiogie,  Parasitenkunde  und  Inlektionskrankheiten,  Abteilung  2,  vol.  8,  No.  11,  pp. 
339-342.  Jena,  Mnr.  13,  1902. 

*Rogers,  I/.  A.  The  bacteria  of  pasteurized  and  unpasteurized  milk  under  laboratory  conditions.  United 
States  Department  of  Agriculture,  Bureau  of  Animal  Industry,  Bulletin  73.  Washington,  1905. 

'Marshall,  C.  E.  Pasteurization  of  milk.  Michigan  Agricultural  Experiment  Station,  Bulletin  147. 
Agricultural  College,  August,  1897. 

4  Ma//,  P.  Pasteurization  du  lait  destin6  Ma  consommation.  I/Industric  Laitiferc,  vol.  32,  No.  8,  pp. 
121-127.  Paris,  Feb.  24,  1907. 

9 
67796°— Bull.  161—13 2 


10  BACTERIA  WHICH   SURVIVE   PASTEURIZATION. 

were  destroyed  by  five  minutes'  heating  at  temperatures  between 
55°  and  65°  C.  (131°  and  149°  F.),  but  that  lactic  acid  bacteria  existed 
which  resist  five  minutes'  heating  at  75°  C.  (167°  F.).  When  milk 
was  pasteurized  in  bottles  for  60  minutes  at  65°  C.  (149°  F.),  Gerber  l 
and  Weiske  found  that  the  bacteria  which  resisted  belonged  for  the 
most  part  to  the  inoffensive  lactic  bacilli.  An  investigation  of  com- 
mercially pasteurized  milk  in  this  country  carried  on  by  the  authors 2 
showed  the  presence  of  high-temperature  resisting  lactic-acid  bacteria, 
the  thermal  death  point  of  one  of  which  was  77.8°  C.  (172°  F.)  with 
a  10  minutes'  exposure  and  75.6°  C.  (168°  F.)  with  a  30  minutes' 
heating.  It  was  further  found  that  commercially  pasteurized  milk 
always  soured.  Kohler3  and  Tonney,  after  studying  pasteurized 
milk  in  Chicago,  concluded  that  reinfection  was  the  cause  of  the  sour- 
ing of  heated  milk. 

It  is  evident  that  the  knowledge  of  the  bacteria  which  actually 
survive  pasteurization  is  very  limited. 

OBJECTS   OF  THIS  INVESTIGATION. 

The  general  object  of  this  investigation  was  to  study  quantitatively 
and  qualitatively  the  bacteria  which  survive  pasteurization  under 
conditions  which  excluded  any  possibility  of  recontamination  of  the 
milk  after  pasteurization. 

The  special  objects  were  as  foUows: 

1.  To  ascertain  the  average  temperature  of  pasteurization  in  both 
the  "holder"  and  "flash"  process  used  throughout  the  country. 

2.  To  determine  the  effect  of  various  pasteurizing  temperatures 
for  one-half  hour's  exposure  upon  the  bacteria  in  different  grades  of 
milk. 

3.  To  study  the  effect  of  holding  periods  longer  than  one-half  hour 
during  pasteurization. 

4.  To  determine  the  effect  of  sudden  cooling  on  bacteria  after 
pasteurization. 

5.  To  study  quantitatively  the  bacterial  groups  which  survive 
pasteurization  at  various  temperatures. 

6.  To  trace  the  development  of  the  various  bacterial  groups  in 
pasteurized  milk  held  at  different  temperatures. 

7.  To  study  qualitatively  the  bacteria  which  survive  pasteurization. 

i  Gerber,  N.,  and  Weiske,  P.    Pasteurisation  des  flacons  dans  la  grande  Industrie  (pasteurisation  avec 
agitation).    Revue  Gen6raledu  Lait,  vol.  2,  No.  8,  pp.  169-177.    Lierre,  Jan.  30, 1903. 

*  Ayers,  S.  Henry,  and  Johnson,  W.  T.,  jr.    The  bacteriology  of  commercially  pasteurized  and  raw 
market  milk.    United  States  Department  of  Agriculture,  Bureau  of  Animal  Industry,  Bulletin  126. 
Washington,  1910. 

*  Kohler,  Gottfried  and  Tonney,  F.  O.    The  control  of  pasteurization.    Journal  of  the  American  Medical 
Association,  vol.  5«,  No.  10,  pp.  713-718.    Chicago,  Mar.  11, 1911. 


METHODS  EMPLOYED.  11 

METHODS. 

In  this  investigation  the  technique  of  plating  and  the  preparation 
of  beef  infusion  agar  with  a  reaction  of  1.5  (Fuller's  scale)  was  carried 
out  according  to  the  recommendations  of  the  committee  of  standard 
methods  for  the  bacteriological  analyses  of  milk.1  In  the  prepara- 
tion of  fermentation  bouillons  and  litmus  lactose  gelatin,  Liebig's 
beef  extract  was  used  as  a  basis. 

Fermentation  bouillons  were  prepared  by  dissolving  4  grams  of 
Liebig's  beef  extract  and  10  grams  of  Witte's  peptone  in  a  liter  of 
water  and  correcting  reaction  to  0.0  (Fuller's  scale).  The  broth 
was  then  brought  up  to  the  sterilizing  temperature  for  five  minutes 
and  filtered.  When  a  clear  filtrate  was  obtained,  0.5  per  cent  potas- 
sium dibasic  phosphate  and  1  per  cent  of  the  substance  to  be  fer- 
mented was  dissolved  in  it.  The  following  substances  were  used 
to  test  the  fermenting  ability  of  the  bacteria: 

Sugars:  Dextrose,  galactose,  lactose,  saccharose,  raffinose.  Alco- 
hols: Mannite,  glycerin.  Starches:  Inulin,  wheat  starch.  Glucoside: 
Salicin. 

The  ability  of  the  bacteria  to  reduce  nitrates  to  nitrites  was  shown 
by  growing  the  organisms  in  a  bouillon  composed  of  0.1  per  cent 
Witte's  peptone  and  0.02  per  cent  potassium  nitrate  (nitrite-free) 
for  14  days  at  30°  C.  (86°  F.)  and  then  testing  for  the  presence  of 
nitrites.  The  liquefaction  of  gelatin  by  an  organism  was  deter- 
mined by  inoculating  with  a  drop  of  milk  culture  on  the  surface  of 
the  tube  of  medium  and  incubating  for  30  days  at  18°  C.  (64.4°  F.) 
and  then  measuring  the  depth  of  liquefaction. 

Special  media. — For  ordinary  routine  plating  of  a  milk  sample,  and 
especially  for  the  determination  of  the  peptonizing  bacteria,  a  casein 
agar  was  devised,2  consisting  of  dissolved  chemically  pure  casein 
and  agar,  with  a  final  reaction  slightly  acid.  In  isolating  gas  formers, 
Smith  tubes  containing  lactose  peptone  bile  3  and  dextrose  liver  broth  * 
were  used. 

All  samples  of  milk  were  pasteurized  in  sterile  flasks  in  the  labora- 
tory. At  all  the  temperatures  except  60°  C.  (140°  F.)  and  65.6°  C. 
(150°  F.),  800  cubic  centimeters  of  milk  were  heated.  During  the 
few  experiments  at  those  temperatures  only  100  cubic  centim- 
eters of  milk  were  used.  In  all  the  experiments  the  period  of 
heating  was  30  minutes  from  the  time  the  pasteurizing  temperature 

1  Report  of  the  Committee  on  Standard  Methods  of  Bacterial  Milk  Analysis.  American  Journal  of 
Public  Hygiene,  new  series,  vol.  6,  No.  2,  pp.  315-345.  Columbus,  Ohio,  May,  1910. 

*  Ayers,  S.  Henry.  Casein  media  adapted  to  determining  bacteria  In  milk.  United  States  Department 
of  Agriculture,  Bureau  of  Animal  Industry,  Twenty-eighth  Annual  Report,  1911  (in  press). 

'  Jackson,  Daniel  D.  A  new  solution  for  the  presumptive  test  for  Bacillus  coll.  Biological  Studies  of 
Pupils  of  W.  T.  Sedgwick,  pp.  292-299.  Boston,  1906. 

1  Jackson,  D.D.,  and  Muer.T.C.  Liverbroth.  A  medium  for  the  determination  of  gas-forming  bacteria 
in  water  and  sewage.  Journal  of  the  American  Public  Health  Association,  vol.  1,  No.  12,  pp.  927-929. 
Urbana,  111.,  December,  1911. 


12  BACTERIA  WHICH    SURVIVE   PASTEURIZATION. 

was  reached.  The  methods  employed  throughout  this  investiga- 
tion make  the  results  comparable  with  those  obtained  by  pasteuri- 
zation in  sealed  bottles. 

TEMPERATURES  USED  THROUGHOUT  THE  COUNTRY. 

It  was  necessary  to  ascertain  the  temperatures  most  universally 
used  at  milk  plants  in  this  country  in  order  that  an  average  tempera- 
ture might  be  employed  in  these  experiments.  Circular  letters 
were  therefore  sent  to  pasteurizing  plants  in  nearly  aU  cities  with 
a  population  of  over  25,000.  Replies  showed  that  the  average  tem- 
perature with  the  "holder"  process  was  62.8°  C.  (145°  F.).  With 
the  "flash"  process  71.1°  C.  (160°  F.)  was  about  the  average.  The 
reports  from  219  milk  plants  which  pasteurized  showed  that  75  used 
the  "holder"  process  and  144  the  "flash"  process. 

Assuming  that  the  correct  temperatures  for  pasteurization  are 
from  60°  to  65.6°  C.  (140°  to  150°  F.)  with  the  "holder"  process,  then 
62  of  the  75  pasteurized  at  the  proper  temperature,  one  used  a  tem- 
perature too  low,  54.4°  to  57.2°  C.  (130°  to  135°  F.),  and  12  too  high, 
ranging  from  66.7°  to  76.7°  C.  (152°  to  170°  F.).  With  the  "flash * 
process  the  correct  temperature  was  assumed  to  be  71.1°C.  (160°  F.). 
As  there  is  usually  a  variation  in  the  range  of  temperature  during 
pasteurization,  the  maximum  and  the  minimum  given  by  each  plant 
have  been  averaged.  When  the  average  was  71.1°  C.  (160°  F.)  it 
was  assumed  that  the  proper  temperature  was  used.  It  was  found 
that  of  144  plants  using  the  "flash"  process  only  61  used  the  correct 
temperature,  61  pasteurized  too  low  and  22  too  high.  The  low  tem- 
peratures ran  down  to  60°  C.  (140°  F.),  the  high  temperatures  up  to 
82.2°  C.  (180°  F.).  These  figures  show  a  decided  lack  of  uniformity 
of  temperatures  used  in  pasteurization.  The  temperatures  below 
60°  C.  (140°  F.)  with  the  "holder"  process  render  no  protection  as 
far  as  the  destruction  of  pathogenic  organisms  is  concerned,  while 
those  above  65.6°  C.  (150°  F.)  only  increase  the  cost  of  pasteurization 
and  tend  to  reduce  the  cream  line.  The  same  is  true  when  using  the 
"flash"  process  below  or  above  temperatures  ranging  from  71.1°  to 
73.9°  C.  (160°  to  165°  F.). 

As  pasteurization  is  practiced,  the  milk  might  have  been  heated 
from  1  minute  at  60°  C.  (140°  F.)  to  30  minutes  at  76.7°  C.  (170°  F.) 
and  it  would  all  be  known  as  pasteurized  milk.  This  lack  of  uni- 
formity is  undoubtedly  due  to  a  misunderstanding  of  the  effects  of 
heat  on  the  bacterial  flora  of  milk. 


BACTEKIAL    REDUCTIONS. 


13 


BACTERIAL     REDUCTIONS     BY     LABORATORY    PASTEURIZATION, 
USING  THE    "HOLDER"   PROCESS. 

Since  regulations  often  require  a  definite  percentage  bacterial 
reduction  and  as  the  efficiency  of  pasteurizers  is  based  on  the  same 
figures,  it  seemed  advisable  to  study  the  quantitative  bacterial  reduc- 
tions under  exact  laboratory  conditions. 

BACTERIAL  REDUCTIONS   AT  60°  C.(l40°  F.)    AND   65.6°  C.  (150°  F.). 

In  these  experiments  the  milk  was  heated  in  sterile  flasks  in  a 
water  bath  at  a  temperature  of  60°  C.  (140°  F.)  and  65.6°  C.  (150°  F.) 
for  a  period  of  30  minutes.  The  temperature  recorded  by  a  ther- 
mometer in  the  milk  was  maintained  for  the  full  30  minutes.  Twelve 
samples  of  milk  from  dairy  X  were  used.  Each  was  divided  and  a 
portion  pasteurized  at  both  temperatures.  The  counts  were  made 
on  litmus  lactose  gelatin  incubated  at  18°  C.  (64.4°  F.)  for  six  days. 
Table  1  shows  the  total  counts  and  efficiency  of  the  process  as  deter- 
mined by  the  percentage  bacterial  reduction.  An  examination  of 
the  table  shows  that  the  raw  milk  was  of  poor  quality,  having  a  high 
bacterial  count.  The  samples  pasteurized  at  60°  C.  (140°  F.)  showed 
a  high  count,  while  the  same  samples  at  65.6°  C.  (150°  F.)  showed  a 
low  count,  as  a  rule.  The  effect  of  the  few  degrees  of  heat  may  be 
plainly  seen.  At  the  lower  temperature  9  out  of  the  12  samples 
showed  a  reduction  of  less  than  99  per  cent,  while  at  the  higher 
temperature  only  4  showed  less  than  a  99  per  cent  reduction. 

TABLE  1. — Percentage  reduction  of  bacteria  by  laboratory  pasteurization,  using  the  "holder" 
process — Raw  milk  obtained  from  dairy  X. 


Pasteurized  at  60°  C. 

Pasteurized  at 

Raw  milk. 

(140°    F.)    for    30 

65.6°  C.  (150°  F.) 

minutes. 

for  30  minutes. 

Sample 

No. 

Total  count. 

Total 
count. 

Percent- 
age re- 
duction. 

Total 
count. 

Percent- 
age re- 
duction. 

1 

33,900,000 

200,000 

99.41 

9,800 

99.97 

2 

4,900,000 

118,000 

97.59 

12,300 

99.74 

3 

5,050,000 

130,  000 

97.45 

25,100 

99.50 

4 

850,000 

32,000 

96.23 

25,500 

97.00 

5 

6,  100,  000 

20,200 

99.  06 

12,100 

99.80 

6 

8,400,000 

23,000 

99.72 

12,600 

99.85 

7 

1,670,000 

284,000 

82.99 

72,000 

95.68 

8 

4,100,000 

74,000 

98.19 

28,500 

99.30 

9 

1,120,000 

66,000 

95.00 

19,900 

98.22 

10 

4,800,000 

153,000 

96.81 

19,800 

99.58 

11 

4,400.000 

200,  000 

95.31 

09,000 

98.43 

12 

12,300,000 

19(1,000 

98.41 

40,000 

99.67 

The  effect  of  pasteurization  on  the  bacterial  content  of  another 
series  of  12  samples  from  dairy  Y  was  studied  in  the  same  way. 
The  results  are  shown  in  Table  2.  It  may  be  seen  that  the  milk 
was  of  far  better  quality  than  that  from  dairy  X.  The  counts  of 


14 


BACTEKIA   WHICH    SURVIVE   PASTEURIZATION. 


milk  pasteurized  at  60°  C.  (140°  F.)  were  low  and  still  lower  at  the 
higher  temperature.  In  spite  of  the  low  counts  the  percentage 
bacterial  reductions  were  not  so  great  as  when  a  poor  grade  of  milk 
was  pasteurized  as  shown  in  Table  1.  Eleven  out  of  twelve  samples 
showed  a  reduction  of  less  than  99  per  cent  at  60°  C.  (140°  F.).  At 
65.6°  C.  (150°  F.)  7  out  of  12  showed  a  bacterial  reduction  of  less 
than  99  per  cent.  A  comparison  of  Tables  1  and  2  show  that  the 
bacterial  content  after  pasteurization  of  the  better  grade  of  milk 
was  far  lower  than  that  of  milk  from  dairy  X,  which  contained  a 
large  number  of  bacteria  before  heating.  If,  however,  the  efficiency 
of  the  process  is  studied,  the  higher  percentage  bacterial  reductions 
will  be  found  in  Table  1,  where  the  milk  was  of  poor  quality.  It  is 
very  evident  that  the  percentage  bacterial  reduction  is  of  no  value 
in  determining  the  quality  of  pasteurized  milk.  Compare,  for 
example,  sample  No.  1  in  Table  1  with  sample  No.  3  in  Table  2.  In 
the  first  case,  the  raw  milk  contained  33,900,000  bacteria  per  cubic 
centimeter  and  after  pasteurization  200,000  per  cubic  centimeter, 
which  is  a  percentage  reduction  of  99.41  per  cent.  In  the  second 
case,  raw  milk,  128,000  bacteria  per  cubic  centimeter  and  after 
pasteurization  13,900  per  cubic  centimeter,  which  is  a  reduction  of 
only  89.14  per  cent.  A  99.41  per  cent  reduction  against  89.14  per 
cent,  and  yet  the  pasteurized  milk  in  which  only  89.14  per  cent  of 
the  bacteria  were  destroyed  was  far  superior  in  its  bacterial  content. 

TABLE  2. — Percentage  reduction  of  bacteria  by  laboratory  pasteurization,  using  the  ' '  holder  " 
process — Raw  milk  obtained  from  dairy  Y. 


Pasteurized  at  60°  C. 

Pasteurized  at 

Raw  milk. 

(140°     F.)    for    30 

65.6°  C.  (150J  F.) 

minutes. 

for  30  minutes. 

Sample 

No. 

Total  count. 

Total 
count. 

Percent- 
age re- 
duction. 

Total 
count. 

Percent- 
age re- 
duction. 

1 

2,500,000 

27,600 

98.89 

4,210 

99.83 

2 

1,090,000 

13,100 

98.79 

2,320 

99.78 

3 

128,000 

13,900 

89.14 

5,670 

95.  57.-. 

4 

149,000 

7,500 

94.96 

3,100 

97.91 

5 

COO,  000 

16,400 

97.26 

11,100 

98.15 

6 

119,000 

2,530 

97.87 

1,020 

99.14 

7 

168,000 

1,890 

98.87 

3,230 

98.07 

8 

84,000 

2,600 

96.90 

2,720 

96.76 

9 

ICO,  000 

5,110 

96.80 

2,010 

98.74 

10 

590,000 

13,200 

97.76 

7,100 

98.79 

11 

2,900,000 

28,700 

99.01 

20,400 

99.29 

12 

1,170,000 

34,100 

97.08 

3,560 

99.69 

The  uselessness  of  figures  in  expressing  percentage  reductions  is 
further  shown  in  Table  3.  The  bacterial  content  of  12  samples  of 
milk  before  and  after  pasteurization  has  been  averaged,  two  grades 
of  milk  having  been  used.  While  the  percentage  reduction  is  approxi- 
mately the  same  at  60°  C.  (140°  F.)  the  average  bacterial  counts  vary 


BACTEKIAL   REDUCTIONS. 


15 


widely,  one  being  124,000  per  cubic  centimeter,  the  other  only  13,900 
per  cubic  centimeter.  At  65.6°  C.  (150°  F.)  while  the  percentage 
reduction  is  increased  to  98  per  cent,  the  bacterial  content  holds  the 
same  relation  as  at  the  lower  temperature. 

TABLE  3. — Comparison  of  the  average  bacterial  content  of  two  grades  of  milk  and  percentage 
reduction  in  bacteria  by  pasteurization  at  60°  C.  (140°  F.)  and  65.6°  C.  (150°  F.). 

AVERAGE  OF  12  SAMPLES  IN  EACH  GRADE. 


Grade. 

Raw  milk. 

Pasteurized  at60°C. 
(140°  F.)  30  minutes. 

Pasteurized  at  65.6°  C. 
(150°  F.)  30  minutes. 

Bacteria 
per  cubic 
centimeter. 

Bacteria 
per  cubic 
centimeter. 

Percent- 
age re- 
duction. 

Bacteria 
per  cubic 
centimeter. 

Percent- 
age re- 
duction. 

Grade  1  
Grade  2  

7,300,000 
804,000 

124,000 
13,900 

96.39 
96.95 

28,900 
5,500 

98.72 
98.40 

BACTEKIAL   REDUCTION   AT   62.8°   C.    (l45°  F.). 

Another  series  of  28  samples  of  milk  was  studied  in  a  similar  man- 
ner. The  results  are  shown  in  Table  4.  In  this  series  a  tempera- 
ture of  62.8°  C.  (145°  F.)  was  used  for  a  period  of  30  minutes.  All  of 
the  samples  of  raw  milk  contained  over  1 ,000,000  bacteria  per  cubic 
centimeter.  The  bacterial  counts  were  made  on  infusion  agar,  incu- 
bated at  30°  C.  (86°  F.)  for  six  days.  It  may  be  seen  from  the  table 
that  the  percentage  reductions  were  high,  only  3  out  of  28  being  less 
than  99  per  cent;  however,  13  of  the  pasteurized  samples  contained 
over  20,000  bacteria  per  cubic  centimeter. 

TABLE  4. — Efficiency  of  the'1  holder"  process  of  pasteurization  under  laboratory  conditions, 
using  raw  milk  with  a  bacterial  content  over  1,000,000  per  cubic  centimeter. 


Pasteurized  at  62.8° 

Pasteurized  at  62.8° 

Raw  milk. 

C.  (145°  F.)  for  30 

Raw  milk. 

C.  (145°  F.)  for  30 

minutes. 

minutes. 

>;ITTI  !(!•' 

No 

Bacteria 
per  cubic 
centimeter. 

Bacteria 
per  cu- 
bic cen- 
timeter. 

Percent- 
age reduc- 
tion. 

Surnplc 
No. 

Bacteria 
per  cubic 
centimeter. 

Bacteria 
per  cu- 
bic centi- 
meter. 

Percent- 
age reduc- 
tion. 

1 

16,800,000 

85,000 

99.49 

15 

12,200,000 

33,300 

99.72 

2 

91,000,000 

14,400 

99.98 

10 

28,  200,  000 

127,000 

99.54 

3 

15,900,000 

18,200 

99.88 

17 

6,480,000 

2,400 

99.% 

4 

90,000,000 

5,800 

99.99 

18 

4,540,000 

9,500 

99.97 

6 

19,800,000 

41,000 

99.79 

19 

2,460,000 

13,600 

99.44 

6 

105,000,000 

17,500 

99.98 

20 

4,900,000 

9,900 

99.79 

7 

47,300,000 

8,900 

99.98 

21 

1,060,000 

1,010 

99.90 

8 

12,400,000 

9,700 

99.92 

22 

2,790,000 

8,800 

99.68 

9 

8,600,000 

15,000 

99.82 

23 

5,400,000 

36,600 

99.32 

10 

17,800,000 

8,900 

99.95 

24 

6,060,000 

30,100 

99.50 

11 

18,900,000 

41,800 

99.97 

25 

1,140,000 

1,560 

99.86 

12 

15,100,000 

39,700 

99.74 

26 

14,600,000 

468,000 

96.79 

13 

22,000,000 

20,400 

99.90 

27 

1,440,000 

22,900 

98.41 

14 

98,000,000 

28,200 

99.97 

28 

1,780,000 

26,500 

98.51 

BACTERIA    WHICH    SURVIVE   PASTEURIZATION. 


The  results  of  similar  experiments  using  a  series  of  24  samples  of 
clean  milk  are  shown  in  Table  5.  The  bacterial  counts  of  the  pasteur- 
ized milk  were  very  low  as  a  rule  and  yet  14  of  the  24  samples  showed 
a  percentage  bacterial  reduction  of  less  than  99  per  cent.  Sample  17 
contained,  before  heating,  only  119,000  bacteria  per  cubic  centimeter; 
after  the  process  the  bacterial  content  was  20,800,  a  reduction  of  only 
82.52  per  cent.  This  sample  evidently  contained  a  high  percentage  of 
resistant  bacteria  which  were  able  to  withstand  the  process  of  pasteur- 
ization. From  these  results  it  is  apparent  that  regulations  of  boards  of 
health  calling  for  a  99  per  cent  reduction  of  bacteria  in  commercial  pas- 
teurization are  of  no  value.  As  a  general  rule,  it  may  be  said  that  the 
higher  the  bacteria  hi  raw  milk  the  greater  the  percentage  reduction 
through  pasteurization.  When  the  bacterial  content  of  milk  is  low  it  is 
almost  always  impossible  to  obtain  a  99  per  cent  reduction  of  the  bacte- 
ria. It  is  also  evident  that  bacterial  standards  for  pasteurized  milk  must 
not  be  set  below  the  limit  which  it  is  theoretically  possible  to  obtain. 
When  pasteurizing  in  the  laboratory  for  a  period  of  30  minutes  at 
62.8°  C.  (145°  F.)  under  conditions  which  permitted  no  reinfection 
of  the  milk,  the  bacterial  content  of  pasteurized  milk  was  by  no  means 
uniform.  Sometimes  the  counts  were  high  and  sometimes  low  and 
yet  the  maintenance  of  the  proper  temperature  of  pasteurization 
would  have  assured  in  all  cases  the  same  degree  of  protection  from 
pathogenic  organisms. 

TABLE  5. — Efficiency  of  the  ' '  holder  "  process  of  pasteurization  under  laboratory  conditions, 
using  raw  milk  with  a  bacterial  content  less  than  1,000,000  per  cubic  centimeter. 


Pasteurized  at  62.8° 

Pasteurized  at  62.8° 

Raw  milk. 

C   (145°  F.)  for  30 

Raw  milk. 

C.  (145°  F.)  for  30 

minutes. 

minutes. 

Sample 

Sample 

No. 

Bacteria 
per  cubic 
centimeter. 

Bacteria 
per  cu- 
bic cen- 
timeter. 

Percent- 
age reduc- 
tion. 

No. 

Bacteria 
per  cubic 
centimeter. 

Bacteria 
per  cu- 
bic cen- 
timeter. 

Percent- 
age reduc- 
tion. 

1 

372,000 

910 

99.75 

13 

540,000 

11,800 

97.81 

2 

434,000 

2,330 

99.46 

14 

970,000 

46,000 

95.25 

3 

540,000 

4,890 

99.09 

15 

162,000 

8,400 

94.81 

4 

410,000 

1,170 

99.71 

16 

118,000 

9,000 

92.37 

5 

127,000 

80 

99.93 

17 

119,000 

20.800 

82.52 

6 

143,000 

765 

99.46 

18 

137,000 

3,100 

97.73 

7 

9,300 

90 

99.03 

19 

15,600 

300 

98.07 

8 

18,100 

180 

99.00 

20 

9,800 

100 

98.97 

9 

19,900 

62 

99.68 

21 

18,700 

1,340 

92.83 

10 

5,300 

34 

99.35 

22 

25,900 

830 

96.79 

11 

134,000 

7,500 

94.40 

23 

15,300 

334 

97.81 

12 

300.000 

7,600 

97.47 

24 

9,900 

320 

96.76 

THE    CONTROL   OF   PASTEURIZATION. 

The  control  of  the  process  of  pasteurization  should  be  by  bacterial 
limits  for  the  milk  which  is  to  be  used,  together  with  supervision  for 
the  maintenance  of  the  proper  pasteurizing  temperature  and  protec- 


EFFECT    OF   LONG   HEATING    ON   BACTEEIA. 


17 


7S000 


70,000 


tion  against  reinfection.  The  bacterial  content  of  the  milk  will  then 
consist  only  of  those  organisms  which  are  able  to  withstand  the  tem- 
perature in  use. 

EFFECT   OF   LONG  HEATING   ON  THE   BACTERIA   IN  MILK. 

In  some  milk  plants  a  holding  period  longer  than  half  an  hour  is 
employed.  It  seems  to  be  believed  that  an  increased  holding  period 
produces  a  greater  bacterial  reduction.  Several  experiments  were 
made  at  different  tem- 
peratures and  for  dif- 
ferent periods  in  order  to 
gain  more  information 
on  this  point. 

THREE      HOURS'     PAS- 
TEURIZATION. 

In  order  to  determine 
the  effect  of  a  long  pe- 
riod of  pasteurization, 
milk  was  heated  in  ster- 
ile flasks  at54.4°C.(130° 
F.),  57.2°  C.  (135°  F.), 
and  60°  C.  (140°  F.),  and 
plated  every  half  hour 
for  three  hours.  The 
bacterial  counts  are 
shown  in  Table  6.  One 
sample  of  milk  was  di- 
vided and  a  portion 
heated  at  the  two  lower 
temperatures,  so  the 
bacterial  content  of  the 
raw  milk  was  the  same. 
The  sample  of  milk 
heated  at  60°  C.  (140°  F.)  contained  approximately  the  same  number 
of  bacteria  per  cubic  centimeter  and  was  from  the  same  dairy,  so  it 
is  probably  fair  to  compare  the  results.  After  one-half  hour's  heat- 
ing a  much  greater  reduction  was  observed  where  the  milk  was  heated 
at  60°  C.  (140°  F.).  At  the  end  of  one  hour's  heating  the  bacterial 
content  of  the  milk  were  more  nearly  alike  and  continued  to  show  a 
gradual  reduction  throughout  the  three  hours  of  heating.  The  final 
examination  showed  that  the  bacterial  content  was  approximately 
the  same  in  all  the  samples  of  milk.  The  bacterial  reductions  are 
shown  better  by  the  curves  in  figure  1 .  It  is  evident  that  long  heating 
67796°— Bull.  161—13 3 


FIG.  1.— Bacterial  reduction  during  pasteurization  for  three  hours 
at  54.4°  C.  (130°  F.),  57.2°  C.  (135°  F.),  and  60°  C.  (140°  F.). 


18 


BACTERIA   WHICH   SURVIVE   PASTEURIZATION. 


at  54.4°  C  (130°  F.)  and  57.2°  C.  (135°  F.)  is  of  more  value  than  at 
60°  C.  (140°  F.),  as  shown  by  curves  A,  B,  and  C,  respectively.  From 
a  comparison  of  curves  A  and  C  it  seems  that  one  and  a  half  hours' 
heating  at  54.4°  C.  (130°  F.)  will  produce  as  great  a  bacterial  reduc- 
tion as  one-half  hour's  heating  at  60°  C.  (140°  F.).  It  must  not  be 
assumed,  however,  that  one  and  one-half  hours'  heating  at  54.4°  C. 
(130°  F.)  will  destroy  pathogenic  organisms  as  surely  as  will  one- 
half  hour's  heating  at  60°  C.  (140°  F.).  That  point  can  only  be 
determined  by  experiment. 

TABLE  6. — Bacterial  reduction  during  pasteurization  for  3  hours  at  54.4°  C.  (130°  F.), 
57.2°  C.  (135°  F.),  and  60°  C.  (140°  F.). 


Temperature 
of 
pasteuriza- 
tion. 

Bacteria  per 
cubic  centi- 
meter in 
raw  milk. 

Bacteria  per  cubic  centimeter  in  milk  pasteurized  for  — 

1  hour. 

1  hour. 

1J  hours. 

2  hours. 

2J  hours. 

3  hours. 

•C.  °F. 
54.4    130 
57.2    135 
60.0    140 

2,530,000 
2,530,000 
2,330,000 

75,000 
63,000 
20,800 

27,600 
15,400 
19,400 

19,400 
12,600 
14,100 

13,200 
8,400 
12,400 

12,300 
7,900 
10,300 

9,200 
5,600 
8,700 

SIX    HOURS    PASTEURIZATION. 

A  similar  experiment  was  made,  using  a  six  hours'  period  of  heating 
at  62.8°  C.  (145°  F.).  In  order  to  meet  extreme  conditions  where 
the  effect  of  long  heating  might  show,  a  milk  containing  27,000,000 
bacteria  per  cubic  centimeter  was  selected.  Plates  were  made  every 
half  hour  during  the  period  of  heating.  Table  7  shows  the  results. 

TABLE  7. — Bacterial  reductions  during  pasteurization  for  6  hours  at  62.8°  C.  (145°  F.) 


Length  of 
pasteur- 
ization 
(hours). 

Bacteria  per 
cubic  centi- 
meter. 

Length  of 
pasteur- 
ization 
(hours). 

Bacteria  per 
cubic  centi- 
meter. 

Raw 

27,000,000 

8} 

32,900 

i 

46,000 

4 

31,200 

1 

46,000 

41 

44,000 

IJ 

45,000 

5 

35,700 

2 

65,000 

B4 

39,000 

2V 

62,000 

6 

34,500 

3" 

54,000 

It  may  be  seen  that  there  was  little  difference  in  the  bacterial  reduc- 
tion produced  by  a  half  hour's  heating  and  by  six  hours'  heating. 
There  were  numerous  variations  in  the  numbers  of  bacteria  as  deter- 
mined, but  they  were  within  the  experimental  error,  as  the  flask  of 
heated  milk  could  not  be  thoroughly  shaken.  In  this  experiment  the 
groups  of  bacteria  which  withstood  the  heating  were  determined.  The 
plating  was  done  on  infusion  agar  plates,  which  were  incubated  at 


EFFECT   OF   LONG   HEATING   ON   BACTERIA. 


19 


73 


70 


60 


Z 


\ 


30°  0.  (86°  F.)  for  six  days.  The  plates  were  counted  and  then  each 
colony  picked  from  a  plate  and  inoculated  into  litmus  milk  tubes. 
After  14  days'  incubation  the  milk  cultures  were  examined  and  as 
'a  result  of  the  reactions  shown  the  bacteria  picked  from  the  plates 
were  divided  into  five  groups,  the  acid  forming  and  coagulating,  acid- 
forming,  inert,  alkali-forming,  and  peptonizing  groups.  The  value 
of  this  method  of  determining  bacterial  groups  will  be  discussed  later 
in  this  paper.  Figure  2  shows  the  relations  of  the  groups  after  heat- 
ing one-half  hour,  three  hours,  and  six  hours.  The  acid-coagulating 
and  simple  acid-forming 
groups  composed  the 
largest  portion  of  the 
bacteria  after  a  half 
hour's  heating  and  the 
same  after  six  hours' 
pasteurization.  It  may 
be  seen,  however,  that 
the  percentage  of  the 
acid-coagulating  group 
was  reduced  by  the 
heating  for  six  hours. 
It  seems  probable  that 
the  heating  weakened 
the  activity  of  the  acid- 
forming  bacteria  so  that 
in  the  litmus  milk  tubes 
not  enough  acid  was 
produced  to  coagulate 
the  milk  in  the  14  days' 
incubation  period. 
That  would  result  in  a 
lowering  of  the  percent- 
age of  the  acid-coagulating  group,  while  increasing  at  the  same  time 
the  simple  acid-forming  group.  The  percentage  of  the  alkali  and 
peptonizing  groups  was  extremely  low  throughout  the  experiment. 
In  figure  3  the  group  percentages  have  been  plotted  at  every  half 
hour's  sampling  and  gives  a  more  complete  picture  of  the  changes 
in  the  bacterial  groups.  The  changes  in  the  group  percentages 
between  tliree  and  one-half  and  four  hours  is  undoubtedly  due  to  the 
errors  in  sampling  and  inaccuracy  of  the  method  of  the  bacterial 
group  differentiation. 


25 


/s 


\ 


FIG.  2. — Bacterial  reduction  during  pasteurization  for  six  hours  at 
62.8°  C.  (145°  F.).    Plotted  after  one-half ,  three,  and  six  hours. 


20 


BACTERIA   WHICH   SURVIVE   PASTEURIZATION. 


EFFECT  OF  SUDDEN  COOLING  ON  THE  BACTERIA  IN  PASTEURIZED 

MILK. 

The  sudden  cooling  of  the  hot  milk  after  pasteurization  has  been 
considered  by  many  to  be  an  essential  part  of  the  process.  It  was* 
believed  that  the  sudden  change  from  hot  to  cold  aided  in  the  destruc- 
tion of  the  bacteria. 
In  the  recent  develop- 
ments of  the  process  of 
pasteurization,  how- 
ever, this  opinion  lost 
ground,  although  at  the 
present  time  there  are 
IS  those  who  still  believe 
in  its  value.  In  order 
to  throw  light  on  this 
point  a  few  experi- 
ments were  made  under 
laboratory  conditions. 


pasteurized  in  sterile 
fe  flasks,  then  cooled  by 
running  through  a 
sterile  copper  coil  sur- 
rounded by  brine  into 
another  sterile  flask. 
Table  8  shows  the  results 
of  the  experiments. 
Two  samples  of  milk 
were  pasteurized  at 
62.8°  C.  (145°  F.)  for  30 
minutes,  then  cooled  in 
15  seconds  to  from  1.7°- 
3.9°  C.  (35°-39°  F.). 

FIG.  3.— Bacterial  reduction  during  pasteurization  for  six  hours  at  The  experiment  Was  T6- 
62.8°  C.  (145°  F.).    Plotted  every  half  hour.  .  .     , 

peated,  using  a  period 

of  30  minutes'  heating  at  71.1°  C.  (160°  F.).  The  bacterial  content 
of  the  milk  before  and  after  cooling  was  approximately  the  same,  the 
differences  always  being  within  the  limits  of  experimental  error.  It  is 
evident  from  the  results  shown  in  the  table  that  sudden  cooling  is 
of  no  value  in  causing  a  destruction  of  bacteria,  at  least  not  at  the 
temperatures  used  in  the  experiments. 


BACTERIAL   GROUPS. 


21 


TABLE  8. — Effect  of  sudden  cooling  on  the  bacterial  content  of  pasteurized  milk. 


Pasteurized  at  62.8°  C. 

Pasteurized  at  71.1°  C. 

(145°  F.)  30  minutes. 

(160°  F.)  30  minutes. 

Sample 
No. 

Bacteria 
per  cubic 
centimeter 
in  raw 
milk. 

Sample 
No. 

Bacteria  per 
cubic  centi- 
meter in  raw 
milk. 

Bacteria 
per  cubic 
centimeter 

Bacteria 
per  cubic 
centimeter 

Bacteria 
per  cubic 
centimeter 

Bacteria 
per  cubic 
centimeter 

in  milk 

in  cooled 

in  milk 

in  cooled 

not  cooled. 

milk. 

not  cooled. 

milk. 

1 

186.000 

8,600 

^    8,500 

3 

400.000 

1,880 

1,950 

2 

233,000 

1,470 

2,160 

4 

1,350,000 

1,750 

1,700 

The  value  of  sudden  cooling  then,  lies  in  the  fact  that  milk  after 
pasteurization  is  not  allowed  to  cool  slowly  through  temperatures 
below  37.8°  C.  (100°  F.)  where  a  rapid  development  of  bacteria  might 
occur. 

GROUPS   OF  BACTERIA  WHICH    SURVIVE   PASTEURIZATION. 

The  most  important  feature  of  this  investigation  has  been  the 

determination  of  the  various  groups  of  bacteria  which  survive  the 

process   of   pasteurization.     These  bacteria   form   the   basis   of  the 
bacterial  growth  in  pasteurized  milk. 

GRADES    OF    MILK    STUDIED. 

Since  the  bacterial  groups  in  various  grades  of  milk  vary  with  the 
quality  of  the  milk,  it  was  decided  to  examine  three  grades — one  of 
poor  quality,  one  of  fair  quality,  and  one  of  good  quality.  The 
bacterial  content  of  each  of  the  samples  of  the  three  grades  has  been 
averaged  as  shown  in  Table  9.  With  poor  milk  designated  grade  A, 
the  average  of  20  samples  showed  a  bacterial  content  of  32,950,000 
per  cubic  centimeter.  An  average  of  24  samples  of  grade  B  showed 
3,451 ,000  per  cubic  centimeter,  while  the  average  bacterial  count  of  12 
samples  of  grade  C  milk  showed  24,700  per  cubic  centimeter. 

TABLE  9. — Grades  of  milk  studied. 


Average 
number  of 

Number 

Grade. 

bacteria 
per  cubic 
centimeter. 

samples 
averaged. 

A 

32.9.50.000 

20 

B 

3.451.000 

24 

C 

24,700 

12 

METHODS. 


In  the  determination  of  the  bacterial  groups  a  method  was  employed 
which  may  be  designated  as  the  milk-tube  method.  This  method,  as 
described  earlier  in  this  paper,  consists  in  picking  off  each  colony  on 
the  plate  and  inoculating  it  into  litmus  milk  tubes.  The  milk  tubes 


22 


BACTERIA   WHICH   SURVIVE   PASTEURIZATION. 


were  incubated  for  14  days  at  30°  C.  (86°  F.),  and  the  bacteria  from 
the  plate  were  then  divided  into  groups  according  to  the  reactions 
which  they  produced  in  the  litmus  milk  tubes.  By  using  this  method 
it  was  possible  to  divide  the  bacteria  developing  on  a  plate  into  five 
groups,  namely,  the  acid-forming  and  coagulating,  the  acid-forming, 
the  inert  which  produce  no  change  in  milk,  the  alkali-forming,  and 
the  peptonizing  groups.  The  acid-coagulating  and  the  acid-forming 
groups  are  not  necessarily  composed  of  distinct  organisms,  although 
the  acid-coagulating  group  probably  included  organisms  which 
always  coagulate  milk,  and  in  the  acid  group  there  are  undoubtedly 
bacteria  which  never  coagulate  milk.  The  bacteria  were  divided  by 
the  reaction  in  litmus  milk  after  incubation  for  14  days  at  30°  C. 
(86°  F.).  It  is  impossible  by  the  ordinary  method  of  plating  on 
litmus  lactose  gelatin  to  separate  the  alkali-forming  bacteria  from 
those  whiqji  are  inert  on  gelatin  on  account  of  the  fact  that  colonies 
of  alkali-forming  bacteria  on  litmus  lactose  gelatin  plates  do  not  form 
enough  alkali  to  produce  a  change  of  the  litmus.  The  alkali  and 
inert  groups  must  then  be  classed  together  and  also  include  acid-form- 
ing bacteria  which  do  not  produce  enough  acid  to  give  a  red  coloration 
on  the  plate. 

In  order  to  show  the  value  of  the  tube  method  a  comparison  with 
the  plate  method  was  made.  Four  different  samples  of  milk  were 
used,  a  portion  of  each  being  heated  to  60°  C.  (140°  F.)  and  also 
65.6°  C.  (150°  F.).  The  pasteurized  milk  was  then  plated  on  litmus 
lactose  gelatin  plates  which  were  incubated  at  18°  C.  (64.4°  F.) 
for  six  days.  A  differential  count  was  then  made  of  the  acid-forming, 
alkali  or  inert,  and  the  peptonizing  colonies.  After  counting,  the 
colonies  were  picked  off  and  inoculated  into  litmus  milk  in  order  to 
complete  the  milk-tube  method.  The  results  of  the  experiments  are 
shown  in  Table  10.  It  may  be  plainly  seen  that  the  plate  method 
was  very  inaccurate.  The  percentage  of  the  acid  group  of  bacteria 
was  always  increased  by  the  milk-tube  method  of  differentiation,  and 
the  alkali  or  inert  groups  were  decreased. 

TABLE  10. — Comparison  of  the  milk-tube  and  plate  methods  for  the  determination  of  the 

bacterial  groups  in  milk. 


Sample 
No. 

Temperature 
of  heating. 

Acid  group. 

Alkali  or  inert 
group. 

Peptonizing  group. 

Milk- 
tube 
method. 

Plate 
method. 

Milk- 
tube 
method. 

Plate 
method. 

Milk- 
tube 
method. 

Plate 
method. 

1 
1 
2 
2 
3 
3 
4 
4 

"C.     °F. 
60.0    140 
65.6    150 
60.0    140 
65.6    150 
60.0    140 
65.6    150 
60.0    140 
65.6    150 

Per  cent. 
73.21 
75.68 
62.29 
77.89 
58.39 
94.36 
92.17 
90.64 

Per  cent. 
58.73 
56.96 
25.15 
12.93 
34.35 
45.68 
56.37 
49.41 

Per  cent. 
16.07 
11.01 
35.16 
18.95 
32.83 
4.83 
7.25 
7.20 

Per  cent. 
40.21 
41.79 
72.32 
86.20 
58.77 
52.15 
42.64 
50.58 

Per  cent. 
10.71 
13.30 
2.54 
3.15 
8.75 
.80 
.56 
2.15 

Per  cent. 
1.05 
1.24 
2.52 
.86 
6.87 
2.15 
.98 

BACTERIAL  GROUPS. 


23 


57.68 


Usually  the  peptonizing  group  of  bacteria  was   also   increased. 

The  differences  in  the  differentiation  of  the  bacterial  groups  is 
shown  graphically  in  figure  4.  The  bacterial  flora  of  four  samples  of 
milk  pasteurized  at  60°  C.  (140°  F.)  as  shown  by  averages  and  deter- 
mined by  the  plate  method  consisted  of  43.65  per  cent  of  the  acid 
group,  53.48  per  cent  of  alkali  or  inert  group,  and  2.85  per  cent  of 
the  peptonizing  group.  When  determined  by  the  milk- tube  method 
the  group  percentages  were  changed  to  71.51  per  cent  of  the  acid 
group,  22.83  per  cent  of  the  alkali  or  inert  group,  and  5.64  per  cent  of 
the  peptonizing  group. 

When  milk  was  pas- 
teurizedat65.6°C.  (150° 
F.)  there  were  marked 
differences  in  the  bacte- 
rial flora  when  determ- 
ined by  the  two  methods 
(see  fig.  4).  The  re- 
sults from  four  samples 
were  averaged.  As  de- 
termined by  the  plate 
method  the  bacterial 
flora  consisted  of  41.24 
per  cent  of  the  acid 
group,  57.68  per  cent  of 
the  alkali  or  inert  group, 
and  1 .06  per  cent  of  the 

.    . 

peptonizing  group. 
When  determined  by 
the  milk-tube  method 
the  percentages  were 
changed  to  84.64  per 
cent  of  the  acid  group, 
10.49  per  cent  of  the  alkali  or  inert  group,  and  4.85  per  cent  of  the 
peptonizing  group.  It  is  evident  from  these  experiments  that  the  milk- 
tube  method  was  much  superior  to  the  litmus  lactose  gelatin  plate 
method  of  differentiation.  Its  advantages  may  be  summarized  as 
follows: 

1.  The  milk-tube  method  is  more  accurate  than  the  plate  method 
for  the  differentiation  of  bacteria  into  groups. 

2.  It  is  possible  by  this  method  to  separate  the  alkali-forming 
group  of  bacteria  from  the  inert  group,  which  is  not  possible  on  lit- 
mus lactose  gelatin  plates. 

3.  The  activity  of  the  bacteria  in  the  various  groups  can  to  some 
extent  be  determined  by  their  ability  to  produce  changes  in  litmus 
milk  after  various  lengths  of  incubation. 


FIG.  4. — Comparison  of  the  milk-tube  and  plate  method  for  the 
determination  of  the  bacterial  groups  in  milk. 


24 


BACTERIA   WHICH   SURVIVE   PASTEURIZATION. 


THE   BACTERIAL   GROUPS   IN    GRADE   A   MILK   BEFORE    AND   AFTER   PAS- 
TEURIZATION   FOR   30   MINUTES    AT   62.8°    C.    (145°  F.). 

Throughout  the  study  of  the  bacterial  groups  in  raw  and  pasteur- 
ized milk  the  milk-tube  method  of  differentiation  has  been  used. 
The  plating  was  on  infusion  agar,  which  was  incubated  for  six  days 
at  30°  C.  (86°  F.).  The  milk  used  in  the  first  experiments  was  of 
poor  quality  and  constitutes  grade  A  milk.  Pasteurization  was  per- 
formed in  sterile  flasks 
and  heated  in  a  water 
bath  for  30  minutes  at 
62.8°C.  (145°F.).  The 
percentage  of  the  differ- 
ent bacterial  groups  was 
determined  before  and 
after  pasteurization  of 
the  milk.  In  order  to 
show  the  results  in  the 
most  simple  manner  the 
group  percentages  of  10 
samples  of  raw  milk  and 
12  of  pasteurized  milk 
have  been  averaged  and 
are  shown  graphically 
in  figure  5.  The  aver- 
age of  the. total  acid 
group  in  raw  milk  was 
46.88  per  cent  of  the 


AC/0 
/0.7/ 


ACJD 

GOAGULA77MG 
3GJ7 


\G/.07 


2S.-3/ 


ALKAU 
6.47 


0EP7OM/Z/N6 
/7.3/ 


/7.S/ 


9.06 


9.77 


FIG.  5. — Bacterial  groups  of  grade  A  milk  before  and  after  pasteuri- 
zation for  30  minutes  at  62.8°  C.  (145°  F.). 


total  bacteria.  After 
pasteurization  the  per- 
centage was  increased 
to  79.78  per  cent.  The 
acid  coagulating  group 
was  decreased  from 
36.17  percent  to  17.91  per  cent.  The  inert  group  was  also  decreased  and 
the  alkali  group  slightly  increased,  from  6.47  per  cent  to  9.77  per 
cent,  which  was  due  to  the  averaging  of  one  sample  of  pasteurized  milk 
which  was  exceptionally  high  in  the  percentage  of  the  alkali  formers. 
The  peptonizmg  group  was  decreased  from  17.31  per  cent  in  the  raw 
milk  to  1 .39  per  cent  in  the  pasteurized  milk.  These  results  are  most 
striking  in  so  far  that  they  are  contrary  to  the  most  generally  accepted 
ideas  of  the  effect  of  pasteurization  on  the  bacterial  flora  of  milk. 


BACTERIAL  GROUPS. 


25 


THE    BACTERIAL   GROUPS   IN   GRADE    A   MILK   BEFORE   AND   AFTER   PAS- 
TEURIZATION  FOR   30   MINUTES    AT   71.1°    C.    (160°   F.). 

A  second  set  of  experiments  was  made,  using  grade  A  milk  which 
Was  pasteurized  at  71.1°  C.  (160°  F.)  for  30  minutes  and  examined 
in  the  same  manner  as  before.  The  averaged  results  of  five  samples 
of  raw  and  six  samples  of  pasteurized  milk  are  shown  in  figure  6. 
The  changes  in  the 
bacterial  groups  were 
about  the  same  as  at 
62.8°  C.  (145°  F.). 
"While  the  percentage 

AC/D 

COAGVLAT/A/G 
24.O7 


AC/D 
//.36 


of  the  total  acid  group 
was  higher  than  in  the 
milk  pasteurized  at 
the  lower  tempera- 
tures, it  will  be  seen 
that  the  percentage  of 
the  acid-coagulating 
group  was  smaller. 
The  acid  group  was 
made  up  largely  of 
slow  acid-forming  or- 
ganisms, as  will  be 
shown  later.  The 
alkali  group  was  re- 
duced and  the  pepton- 
izing  group  reduced 
from  16.26  per  cent 
in  the  raw  to  0.53  per 
cent  in  the  pasteurized 
milk. 


\B439 


FIG.  6.— Bacterial  groups  in  grade  A  milk  before  and  after  pasteuriza- 
tion for  30  minutes  at  71.1°  C.  (160°  F.). 


THE   BACTERIAL   GROUPS    IN    GRADE    B   MILK   BEFORE    AND   AFTER   PAS- 
TEURIZATION   FOR   30   MINUTES   AT   62.8°  C.    (145°  F.). 

The  same  experiments  were  repeated,  pasteurizing  a  better  quality 
of  milk,  grade  B,  at  62.8°  C.  (145°  F.).  The  averaged  results  of  17 
samples  of  raw  and  20  samples  of  pasteurized  milk  are  shown  in  figure 
7.  The  total  acid  group  was  increased  from  22.72  per  cent  in  the 
raw  to  66.76  per  cent  in  the  pasteurized  milk.  It  will  be  noticed  that 
the  acid-coagulating  group  was  increased  from  12.98  per  cent  to 
31.89  per  cent  in  the  pasteurized  milk.  The  alkali  and  peptonizing 
groups  were  reduced  from  19.66  per  cent  to  5.63  per  cent  and  from 
14.10  to  3.59  per  cent,  respectively. 
67796°— Bull.  161—13 4 


26 


BACTERIA   WHICH    SURVIVE   PASTEURIZATION. 


AC/D 


AC/D        ( 
COAGULAT/A/G{ 
/2.98 


34.87 


3A&9 


THE    BACTERIAL   GROUPS   IN   GRADE    C   MILK   BEFORE    AND    AFTER   PAS- 
TEURIZATION  FOR   30   MINUTES   AT   62.8°  C.   (145°  F.). 

A  third  grade  of  milk  of  good  quality,  grade  C,  was  studied 
in  the  same  manner  as  the  other  grades.  The  percentage  of  the 
various  bacterial  groups  changed  in  a  similar  manner  as  in  the  other 
experiments.  The  milk  was  pasteurized  for  30  minutes  at  62.8°  C. 
(145°  F.).  Figure  8  shows  the  averaged  results.  It  may  be  seen 
that  again  the  total  acid  group  was  increased  in  percentage  from 
40.70  per  cent  to  73.10  per  cent  of  the  total  bacteria.  The  acid- 
coagulating  group, 
however,  decreased  in 
percentage  from  33.85 
per  cent  to  11.85  per 
cent.  All  the  other 
groups  were  decreased 
in  their  percentage  by 
pasteurization.  These 
results  show  that  in- 
stead of  the  acid-form- 
ing bacteria  being 
destroyed  by  pasteuri- 
zation they  were  ac- 
tually increased  in 
their  percentage  of 
the  total  bacteria  sur- 
24.OO  viving  the  process. 
This  does  not  mean 
that  there  were  greater 
numbers  of  acid-form- 
ing  bacteria  after 
pasteurization  but 
merely  that  a  greater 
percentage  of  the  bac- 
teria in  pasteurized 
milk  were  of  the  acid-forming  group  than  in  the  milk  before  heat- 
ing. The  percentage  of  the  other  groups  was  lowered  by  pasteuri- 
zation. This  is  of  particular  interest  in  the  case  of  the  peptonizing 
group,  which  has  been  believed  to  constitute  the  majority  of  the 
bacteria  which  survive  the  heating  process.  It  must  be  remembered 
that  these  results  apply  for  the  most  part  to  milk  pasteurized  at 
62.8°  C.  (145°  F.),  although  they  hold  for  the  few  samples  studied 
which  were  heated  at  71.1°  C.  (160°  F.).  So  far  as  the  practical  side 
is  concerned,  only  the  results  at  62.8°  C.  (140°  F.)  are  of  value,  for 
the  higher  temperatures  are  seldom  used  in  commercial  work. 


FIG.  7. — Bacterial  groups  in  grade  B  milk  before  and  after  pasteuri- 
zation for  30  minutes  at  62.8°  C.  (145°  F.). 


BACTERIAL   GROUPS. 


27 


THE    BACTERIAL    GROUPS    IN    MILK    PASTEURIZED    AT    HIGH 
TEMPERATURES . 

In  order  to  determine  the  bacteria  which  survive  pasteurization 
at  high  temperatures,  the  bacterial  groups  were  determined  by  the 
milk-tube  method  in  seven  samples  of  milk.  Two  samples  were 
pasteurized  at  76.7°  C,  (170°  F.),  three  at  82.2°  C.  (180°  F.),  one  at 
87.8°  C.  (190°  F.),  and  two  at  93.3°  C.  (200°  F.).  All  were  heated  in 
sterile  flasks  and  the  temperatures  held  for  30  minutes.  The  per- 
centages of  the  bacterial  groups  have  been  averaged  at  each  temper- 
ature and  are  shown 
in  figure  9.  It  will  be 
seen  that  even  at  76.7° 
C.  (170°  F.)  a  large 
percentage  of  the  acid- 
forming  group  was 
found,  the  percentage  33.85 
of  the  total  acid-form- 
ing group,  including 
both  the  acid-coagulat- 
ing and  the  simple  acid- 
forming  group,  being 
80.91  per  cent.  The 
bacteria  of  this  group, 
however,  produced 
acid  very  slowly, which 
differentiates  them 
from  those  which  sur- 
vive at  lower  tempera- 
tures. An  increase  in 
the  percentage  of  the 
peptonizing  group 
over  that  at  the  lower 
temperatures  is  notice- 
able. The  average  per- 
centage of  the  pepton- 
izing group  at  62.8°  C.  (145°  F.)  ranged  from  1.39  per  cent  to  4.47  per 
cent  in  three  grades  of  milk.  At  71 .1  °  C.  (1 60°  F.)  the  percentage  was 
0.53  per  cent,  while  at  76.7°  C.  (1 70°  F.)  it  was  increased  to  7.25  percent. 
The  increase  is  of  course  produced  by  the  greater  destruction  of  the  other 
groups.  At  82. 2°  C.  (1 80°  F.)  a  distinct  change  took  place  in  percentage 
of  the  groups  which  survived  the  heating.  The  acid  group  was  reduced 
greatly,  while  the  peptonizing  group  was  increased  to  68.12  per  cent 
of  the  total  bacteria.  The  alkali  group  comprised  16.08  per  cent  of 
the  bacteria,  which  is  evidently  a  very  high  average.  If  a  large 
number  of  samples  had  been  studied,  the  average  percentage  of  the 


PEPTOWZ/MG 

( 


FIG.  8.— Bacterial  groups  in  grade  C  milk  before  and  after  pasteuriza- 
tion for  30  minutes  at  62.8°  C.  (145°  F.). 


28 


BACTERIA   WHICH    SURVIVE   PASTEURIZATION. 


ACJO 
COA6ULAT/NC 


group  would  have  probably  been  very  low,  since  at  71.1°  C.  (160°  F.) 
and  76.7°  C.  (170°  F.)  only  a  very  small  percentage  survived.  The 
bacterial  groups  at  the  higher  temperatures  were  similar  to  those  at 
82.2°  C.  (180°  F.).  The  almost  total  absence  of  the  acid-coagulating 
group  and  the  low  percentage  of  the  acid  group,  together  with  the 
high  percentage  of  the  peptonizing  group,  is  very  noticeable. 

THE  ACTIVITY  OF  BACTERIA  OF  THE  VARIOUS  GROUPS  ISOLATED 
FROM  RAW  AND  PASTEURIZED  MILK  MEASURED  BY  THEIR 
ABILITY  TO  PRODUCE  CHANGES  IN  LITMUS  MILK  AFTER  DIF- 
FERENT LENGTHS  OF  INCUBATION. 

The  activity  of  the  bacteria  found  in  raw  and  pasteurized  milk  is 
important  since  acid-forming  bacteria  which  coagulates  milk  in  48 
hours  produce  a  more  normal  souring  iij  pasteurized  milk  than  the 

Kxyra.      .^^^^k      sl°w     a°id  -  producing 


forms  which  may  be 
overgrown  by  the  pep- 
tonizing bacteria.  Or- 
ganisms of  the  pep- 
tonizing group  which 
cause  peptonization 
rapidly  are  of  far 
greater  importance 
than  those  which  re- 
quire long  incubation 
to  produce  changes  in 
milk.  During  the  de- 
termination of  the  bac- 
terial groups  by  the 
milk-tube  method  the 
reactions  produced  by 
the  different  cultures  of  bacteria  were  recorded  after  2,  5,  and  14  days' 
incubation.  The  length  of  time  required  to  produce  changes  in  litmus 
milk  served  as  a  measure  of  the  activity  of  the  culture.  It  then  fol- 
lows that  the  changes  in  the  group  relations  as  determined  by  the 
litmus  milk  tube  reactions  gave  a  general  indication  of  the  activity 
of  the  bacteria  which  composed  the  group. 

The  activity  of  bacteria  of  the  various  groups  isolated  from  three 
grades  of  milk  has  been  studied. 


ffPTOMZ/fiiG 


FIG.  9.  —  Bacterial  groups  which  survive  pasteurization  for  30  min- 
utes at  76.7°  C.  (170°  F.),  82.2°  C.  (180°F.),  87.8  C.  (190°  F.),  and 
93.3°  C.  (200°  F.). 


GRADE    A   MILK    BEFORE  AND    AFTER    PASTEURIZATION  AT    62. 8C 
(145°    F.)  FOR    30  MINUTES. 


0. 


The  results  shown  in  Table  1 1  indicate  the  rapidity  of  the  growth  of 
the  bacteria  of  the  various  groups  isolated  from  milk  before  and  after 
pasteurization  at  62.8°  C.  (147>°  F.)  as  measured  by  their  ability  to 
produce  a  change  in  litmus  milk.  As  may  be  noted  from  the  table, 


BACTERIAL    GROUPS. 


29 


plates  were  made  of  infusion  and  casein  agar  and  the  colonies  develop- 
ing on  each,  medium  were  picked  off  and  differentiated  by  the  tube 
method.  The  group  percentages  in  the  table  represent  the  average 
of  a  number  of  samples.  In  general  it  may  be  said  that  casein  agar 
seemed  to  favor  the  growth  of  the  alkali  forming  and  the  peptonizing 
bacteria  so  'that  their  group  percentages  were  usuaUy  higher  when 
determined  on  that  medium.  In  order  to  show  the  results  more 
plainly  the  averages  have  been  plotted  as  shown  in  figure  10.  The 
blocks  in  the  columns  represent  the  bacterial  group  percentages 
determined  by  the  tube  method  after  2,  5,  and  14  days'  incubation  in 
milk.  The  column  at  2  days,  for  example,  shows  the  group  percent, 
ages  determined  by  the  reactions  of  the  colonies  picked  from  an 
infusion  agar  plate. 
The  percentages  were 
determined  by  the  re- 
actions of  the  milk  tubes 
after  2  days'  incubation 
at  30°  C.  (86°  F.).  The 
tubes  were  then  incu- 
bated for  3  days  more 
and  the  group  percent- 


AC/D 
COAGULATWG 


3OM/NUTES 

FIG.  10.— Changes  in  the  bacterial  group  relations  in  grade  A  milk 
when  determined  by  litmus  milk  reactions  after  different  lengths 
of  incubation.  Milk  pasteurised  at  62.8°  C.  (145°  F.)  for  30 
minutes. 


ages  again  determined 
by  the  reactions.  Incu- 
bation was  then  contin- 
ued for  14  days,  when 
the  final  reactions  were 
recorded.  The  figure 
shows  that  the  percent- 
age of  the  total  acid 
group  was  greater  in  raw 
milk  after  two  days' 
incubation  than  in  pas- 
teurized. After  five 
days,  however,  the  total  acid  group  was  higher  in  percentage  in 
pasteurized  milk.  After  14  days  the  total  acid  group  in  pasteurized 
milk  was  much  larger.  This  shows  that  the  bacteria  of  the  acid 
group  in  pasteurized  milk  develop  slower,  or  at  least  produce  acid 
slower,  than  those  in  raw  milk.  Since  the  differentiation  of  the 
groups  was  based  on  the  change  produced  in  litmus  milk  by  the  bac- 
teria, it  may  be  seen  from  the  figure  that  as  a  rule  the  alkali-forming 
bacteria  did  not  develop  enough  alkali  to  change  the  color  of  litmus, 
which  differentiates  them  as  belonging  to  the  alkali  group,  until  the 
fifth  day  of  incubation.  While  the  figure  may  seem  rather  compli- 
cated, a  careful  study  clearly  shows  the  difference  in  the  bacterial 
groups  in  raw  and  pasteurized  milk  based  on  the  rapidity  with  which 
they  produce  changes  in  litmus  milk. 


30 


BACTERTA    WHICH    SURVIVE    PASTEURIZATION. 


TABLE  11. — Changes  in  the  bacterial  group  relations  in  grade  A  milk  when  determined  by 
litmus  milk  reactions  after  different  lengths  of  incubation — Milk  pasteurized  at  62.8°  C. 
(145°  F.)for  80  minutes. 


Bacterial  groups. 

Infusion  agar  plates. 

Casein  agar  plates. 

Raw  milk. 

Pasteurized  milk. 

Raw  milk. 

Pasteurized  milk. 

2 
days. 

days. 

14 
days. 

2 
days. 

5 
days. 

14 
days. 

2 
days. 

5 
days. 

14 
days. 

2 
days. 

5 

days. 

14 
days. 

Acid  coagulating 
Acid  . 

Perot. 
24.  IS 
18.08 
42.94 
1.29 
13.47 

99.96 

Per  ct. 
32.72 
14.64 
33.23 
4.02 
15.37 

Perot. 
36.17 
10.71 
29.31 
6.47 
17.31 

Perct. 
6.01 
19.01 
74.69 
.02 
.26 

Perct. 

9.52 
48.01 
35.13 
6.55 

.77 

Perct. 
17.91 
61.87 
9.06 
9.77 
1.39 

Perct. 
7.89 
15.13 
62.26 
.70 
13.99 

Perct. 
16.96 
14.34 
45.95 
5.69 
17.04 

Per  ct. 
19.51 
12.12 
32.99 
17.58 
17.77 

Perct. 

2.42 
11.63 
85.80 

Perct. 
4.34 
42.17 
48.67 
4.17 
.62 

Perct. 
15.12 
51.95 
15.98 
15.73 
1.20 

Inert  

Alkali 

Peptonizing  
Total  

Number  of  sam- 
ples averaged.  . 

.12 

99.98 

99.97 

99.99 

99.98 

100.00 

99.97 

99.  9S 

99.97 

99.97 

99.97 

99.98 

10 

10 

10 

12 

12 

12 

7 

7 

7 

10 

10 

10 

GRADE   A   MILK   BEFORE    AND  AFTER  PASTEURIZATION   FOR   30  MINUTES 

AT  71.1°  C.  (160°  F.). 

Earlier  in  this  paper  it  was  shown  that  the  percentage  of  the  total 
acid  group  was  higher  in  grade  A  milk  pasteurized  at  71.1°  C.  (160° 
F.)  than  at  62.8°  C.  (145°  F.).  It  is  natural  to  suppose,  then,  that 
milk  pasteurized  at  the  higher  temperatures  would  sour  more  quickly 
than  when  heated  at  the  lower  temperatures.  Such  is  not  the  case, 

however;  first,  because 
the  total  number  of 
bacteria  are  less  in  the 
milk  pasteurized  at 
71.1°C.(160°F.),and, 
second,  because  the 
acid  group  of  bacteria 
is  less  active.  This 
last  reason  is  shown 
by  the  changes  in  the 
bacterial  group  rela- 
tions as  determined  in 
litmus  milk  by  differ- 
ent lengths  of  incuba- 
tion. Table  12  shows 
the  averaged  group 
percentages  deter- 
mined from  infusion 
and  casein  agar  plates. 
The  results  are  shown 
to  better  advantage  in  figure  11.  It  will  be  noticed  that  after  two 
days'  incubation  only  a  small  percentage  of  acid-forming  bacteria 
were  recorded  in  the  pasteurized  milk  and  none  of  the  acid-coagulat- 


FIG.  1 1 . — Changes  in  the  bacterial  group  relations  in  grade  A  milk  when 
determined  by  litmus  milk  reactions  after  different  lengths  of  incu- 
bation. Milk  pasteurized  at  71.1°  C.  (160°  F.)  for  30  minutes. 


BACTERIAL  GEOUPS. 


31 


ing  group.  After  five  days  the  acid  group  was  increased  greatly,  but 
there  was  only  an  extremely  small  percentage  of  the  acid-coagulating 
group.  Even  after  14  days'  incubation  the  reactions  showed  only  a 
small  percentage  of  the  acid-coagulating  group.  In  both  raw  and 
pasteurized  milk  the  slow  growth  of  the  alkali  group  is  shown  by  the 
fact  that  only  a  small  percentage  was  found  in  the  raw  milk  after 
five  days'  incubation  of  the  litmus  milk  tubes,  while  none  was  found 
in  the  pasteurized  milk  until  after  14  days'  incubation. 

TABLE  12. — Changes  in  the  bacterial  group  relations  in  grade  A  milk  when  determined  by 
litmus  milk  reactions  after  different  lengths  of  incubation — Milk  pasteurized  at  71.1°  C. 
(160°  F.)for  30  minutes. 


Bacterial 
groups. 

Infusion  agar  plates. 

Casein  agar  plates. 

Raw  milk. 

Pasteurized  milk. 

Raw  milk. 

Pasteurized  milk. 

2 
days. 

5 
days. 

14 
days. 

2 
days. 

5 
days. 

14 
days. 

2 
days. 

5 
days. 

14 
days. 

2 
days. 

5 
days. 

14 
days. 

Acid  coagulat- 
ine    . 

Perct. 
16.58 

Perct. 
20.22 

Perct. 
24.07 

Perct. 

Perct. 
0.17 

85.29 
14.14 

Perct. 
5.49 
84.59 
8.99 
.40 

Perct. 
12.47 
10.65 
64.30 
.13 

Perct. 
16.49 
6.44 
58.85 
2.43 

Perct. 
17.50 
7.22 
47.28 
10.06 

Perct. 

Perct. 

Perct. 
10.88 
63.44 
24.93 

Acid  

16.35 
52.95 

14.60 
49.51 
.89 

11.36 
43.51 
4.77 

12.03 
87.56 

7.67 
92.26 

70.10 
29.53 

Inert  

Alkali  

Peptonizing  — 
Total  

Number  of  sam- 
ples averaged. 

14.11 

14.76 

16.26 

.39 

.39 

.53 

12.43 

15.77 

17.92 

.06 

.36 

.70 

99.99 

99.98 

99.97 

99.98 

99.99 

100.00 

99.98 

99.98 

99.98 

99.99 

99.99 

99.95 

5 

5 

5 

6 

6 

6 

9 

9 

9 

11 

11 

11 

GRADE    B   MILK   BEFORE  AND  AFTER    PASTEURIZATION   FOR  30  MINUTES 

AT  62.8°   C.  (145°   F.) 

The  changes  in  the  bacterial  group  relations  determined  by  the 
milk-tube  method  after 
various  lengths  of  incu- 
bation showed  a  differ- 
ence from  those  in  grade 
A  milk.  Table  13  shows 
the  averages  of  the  group 
percentages  which  are 
plotted  in  figure  12.  A 
study  of  the  plot  shows 
that  the  percentage  of 
the  total  acid  group  and 
the  acid  -  coagulating 
group  itself  was  greater 
in  the  pasteurized  milk  fvfmw/zwei 
after  the  two  days'  in-  OAKS 

cubation  period  than  in 
the  raw  milk.  In  other 
words,  the  activity  of 
the  acid-producing  or- 
ganisms which  survived 
the  heating  was  as  great  as  those  in  raw  milk.  The  reason  for  the 
difference  in  the  two  grades  of  milk  is  not  apparent. 


a*rs-*z      5     /# 

TUffGSEQfi'f/l.tr 

'C  (/tStj 
7&S 


Fio.  12.— Changes  In  the  bacterial  group  relations  in  grade  B  milk 
when  determined  by  litmus  milk  reactions  after  different  lengths 
of  incubation. 


32 


BACTERIA   WHICH    SURVIVE   PASTEURIZATION. 


TABLE  13. — Changes  in  the  bacterial  group  relations  in  grade  B  milk  when  determined  by 
litmus  milk  reactions  after  different  lengths  of  incubation — Milk  pasteurized  at  62.8°  C. 
(145°  F.)for  30  minutes. 


Bacterial 
groups. 

Infusion  agar  plates. 

Casein  agar  plates. 

Raw  milk. 

Pasteurized  milk. 

Raw  milk. 

Pasteurized  milk. 

2 
days. 

5 
days. 

14 

days. 

2 
days. 

5 
days. 

14 

days. 

2 
days. 

5 
days. 

14 

days. 

2 

days. 

5 
days. 

14 
days. 

Acid  coagulat- 
ing     

Perct. 
6.59 
11.39 
73  45 

Perct. 
9.54 
12.55 
58.04 

Per  ct. 
12.98 
9.74 
43.51 

Per  ct. 
20.05 
11.27 
67.46 

Per  ct. 
23.12 
27.48 
45.02 

Per  ct. 
31.89 
34.87 
24.00 

Perct. 
6.14 
4.67 
81.37 

Per  ct. 
7.66 
5.17 
66.89 

Per  ct. 
8.45 
7.18 
50.09 

Perct. 
10.58 
6.72 
82.30 

Perct. 
12.86 
27.98 
55.84 

Perct. 

18.73 
29.72 
40.63 

Acid  

Inert 

Alkali  
Peptonizing  

Total  

Number  of  sam- 
ples averaged 

.21 

8  35 

7.86 
11  99 

19.66 
14.10 

"Lai 

2.39 
1.98 

5.63 
3.59 

.09 
7.69 

6.53 
13.73 

17.79 
16.  47 

.03 

.35 

2.43 

.87 

7.92 
2.99 

99.99 

99.98 

99.99 

99.99 

99.99 

99.98 

99.96 

99.98 

99.98  i  99.98 

99.98 

99.99 

16 

17 

17 

19 

20 

20 

17 

18 

18 

21 

22 

22 

GRADE    C   MILK   BEFORE  AND  AFTER    PASTEURIZATION  FOR  30  MINUTES 

AT    62.8°   C.    (145°   F.) 

When  grade  C  milk  was  studied  in  the  same  manner,  the  per- 
centage of  the  acid-coagulating  group  in  pasteurized  milk  was  found 
to  be  small  and  the  group  inactive.  The  averaged  results  in  Table  14^ 
and  shown  graphically  in  figure  13,  bring  out  this  point.  It  will  be 
seen  that  the  acid-coagulating  group  percentage  was  very  small  up 
to  the  14  days'  incubation  period.  The  acid  group  also  was  rather 
inactive  as  indicated  by  the  percentages.  A  large  part  of  the  pepton- 
izing  group  developed  slowly,  as  indicated  by  the  increase  in  the 
percentage  of  groups  shown  in  the  various  plots. 

TABLE  14. — Changes  in  the  bacterial  group  relations  in  grade  C  milk  when  determined  by 
litmus  milk  reactions  after  different  lengths  of  incubation — Milk  pasteurized  at  62.8°  C. 
(146°  F.)for  30  minutes. 


Bacterial 
groups. 

Infusion  agar  plates. 

Casein  agar  plates. 

Raw  milk. 

Pasteurized  milk. 

Raw  milk. 

Pasteurized  milk. 

2 

days. 

5 
days. 

14 

days. 

2 
days. 

5 
days. 

14 

days. 

2 
days. 

5 
days. 

14 
days. 

2 

days. 

5 
days. 

14 
days. 

Acid  coagulat- 
ing   

Perct. 
18.10 
15.18 
64.54 
.20 
1.96 

Ptra,. 
31.33 
9.42 
49  44 
1.66 
8.13 

Perct. 
33.85 
6.85 
43.13 
3.33 
12.81 

Perct. 
0.06 
6.82 
91.21 

1.89 

Per  ct. 
0.35 
43.02 
53.46 
.06 
3.09 

Per  ct. 
11.85 
61.25 
22.06 
.36 
4.47 

Perct. 
7.25 
5.59 
84.53 

2.61 

Per  ct. 
9.96 
7.17 
76.09 
1.11 
5.65 

Per  ct. 
9.90 
7.94 
63.79 
5.90 
12.44 

Perct. 
0.36 
1.96 
93.02 

4.65 

Perct. 
0.36 
19.84 
71.53 
.90 
7.35 

Perct. 
3.19 

34.57 
49.74 
2.99 
9.49 

Acid          

Inert    

Alkali  
Peptonizing  — 

Total  

Number  of  sam- 
ples averaged 

99.98 

99.98 

99.97 

99.98 

99.98 

99.99 

99.98 

99.98 

99.97 

99.99 

99.98 

99.98 

12 

12 

12 

11 

11 

11 

9 

9 

9 

11 

11 

11 

It  is  interesting  to  note  the  activity  of  the  total  acid  group  in 
pasteurized  milk  of  grade  B,  and  although  the  average  bacterial  count 
was  high  this  grade  of  milk  more  nearly  represents  the  ordinary  quality 
of  milk  which  is  pasteurized  commercially  than  either  grades  A  or  C. 


PEECENTAGE    OF   ACID   FOEMING   BACTEEIA. 


33 


AC/O 

COAGULAT/HG} 


AC/D 


COMPARISON  OF  THE  PERCENTAGE  OF  ACID-FORMING  BACTERIA 
IN  RAW   AND   PASTEURIZED   MILK. 

As  has  already  been  shown,  the  acid  bacteria  instead  of  being 
destroyed  by  pasteurization  for  30  minutes  at  62.8°  C.  (145°  F.)  are 
actually  increased  in  their  percentage  of  the  total  bacteria.  The 
large  percentage  of  the  / 

acid  group  which  survive      AC/O     } 

the  process  are  shown  in 
the  frequency  curve  in 
figure  14.  The  number 
of  samples  were  plotted 
as  ordinates  and  the  per- 
centage of  the  acid-form- 
ing bacteria  as  abscissae, 
the  ranges  in  percent- 
ages from  0-1,  1-10, 
10-20,  etc.,  being  taken 
as  points.  Thirty-nine 
samples  of  raw  and  42  of 
pasteurized  milk  were  ex- 
amined.  The  samples  of 
milk  were  pasteurized  for 
30  minutes  at  62.8°  C. 

ft  A  n°  T?  '\        T*      '11  K  FlG>  13'~~ chanSes  in  the  bacterial  group  relations  in  grade  Cmilk 

,145    r.}.      It  Will  De  Seen        wnen  determined  by  litmusmilk  reactions  after  different  lengths 

from  the  figure  that  with      of  incubation. 

the  raw  milk  the  peak  of  the  curve  is  at  1-10,  while  one  peak  of  the 

curve  of  pasteurized  milk  is  at  10-20  and  another  at  50-60.     In  other 


20^30 30-40        40-50         SO-SO          6O-7O         TV'SO         BO-OO    9O-VO 
ffff  C£NT  Of  AC/O  FQf?/*f/A/G  BAGTFf?/* 
FIG.  14.— Comparison  of  the  percentage  of  the  acid-forming  bacteria  in  raw  and  pasteurized  milk. 

words,  the  majority  of  the  samples  of  raw  milk  contained  from  1  to  10 
per  cent  of  bacteria  of  the  acid  group.     The  number  of  samples  con- 
67796°— BulU61— 13 5 


34 


BACTEEIA   WHICH    SURVIVE   PASTEURIZATION. 


taming  higher  percentages  gradually  diminished  until  no  sample 
contained  over  80  per  cent.  Among  the  samples  of  the  pasteurized 
milk,  it  may  be  seen  from  the  curve  that  the  majority  of  them 
contained  from  10  to  60  per  cent  of  the  acid  group  and  some  samples 
as  high  as  90  per  cent.  .-' 

COMPARISON  OF  THE  PERCENTAGE   OF  PEPTONIZING  BACTERIA 
IN  RAW   AND   PASTEURIZED   MILK. 

In  the  same  manner  39  samples  of  raw  milk  and  42  samples  of 
milk  pasteurized  for  30  minutes  at  62. 8°  C.  (145°  F.)  have  been  grouped 
according  to  the  percentage  of  peptonizing  bacteria  which  they  con- 
tained. A  comparison  of  the  frequency  curves  in  figure  15  shows 
that  the  pasteurized  milk  contained  a  lower  percentage  of  the  pepton- 
izing group  than  did  the  raw  milk.  A  large  number  of  samples  of 
pasteurized  milk  contained  between  0  and  1  per  cent  of  peptonizing 

bacteria,  while  only  one 
sample  of  raw  milk  con- 
tained that  low  percent- 
age. The  same  number 
of  samples  of  both  raw 
and  pasteurized  milk 
contained  from  1  to  10 
per  cent.  It  will  also 
be  seen  from  the  curve 
that  a  few  samples  of 

i-/o         /o-zo       20-30        30-40       *o-5o    &>-&>    V^f},    raw    anfj    r>ast,PUr- 

f>fff  Cf/VT  Of  PFP7T3M/Z/MG  BACTfff/A 

FIG.  15.— Comparison  of  the  percentage  of  the  peptonizing  bacteria  ized       milk        Contained 
in  raw  and  pasteurized  milk.  ^^  ^^   j  t()  1Q  ^ 

cent  of  the  peptonizing  group.  However,  in  a  comparison  of  the 
raw  and  pasteurized  milk  it  was  found  that  a  larger  number  of 
samples  of  raw  milk  contained  the  high  percentages  of  peptonizers. 

AVERAGE   PERCENTAGES   OF  THE   BACTERIAL   GROUPS  OF   RAW 
MTT/KT  WHICH    SURVIVE    PASTEURIZATION. 

The  effect  of  pasteurization  upon  the  bacteria  in  milk  may  be 
shown  by  a  determination  of  the  percentage  of  the  bacterial  groups  of 
raw  milk  which  are  found  after  heating.  This  percentage  was 
obtained  by  subtracting  the  percentage  reduction  by  pasteurization 
of  the  various  groups  from  100  per  cent.  The  samples  of  raw  and 
pasteurized  milk  were  plated  on  infusion  and  casein  agar  and  the 
percentage  reduction  of  the  various  groups  determined  by  the  milk 
tube  method.  Table  15  shows  the  averaged  results  from  infusion 
and  casein  agar  plates,  using  three  grades  of  milk  for  examination. 


EFFECT   OF   PASTEURIZATION   ON    NUMBER   OF   BACTERIA. 


35 


It  may  be  seen  that  a  greater  percentage  of  the  acid  group  of  bacteria 
in  the  raw  milk  remained  after  heating  than  any  of  the  other  groups. 
As  a  rule  a  smaller  percentage  of  the  peptonizing  group  survived 
than  either  of  the  other  groups.  In  general  the  result  when  casein 
agar  was  used  was  higher,  although  the  reason  is  not  apparent. 

TABLE  15. — Average  percentages  of  the  bacterial  groups  of  raw  milk  which  survive 
pasteurization  by  the  "holder"  process. 


Pasteurization  temper- 
ature. 

Media. 

Bacterial  groups. 

Acid. 

Inert. 

Alkali. 

Pepton- 
izing. 

Grade  A,  62.8°  C.  (145°  F.).. 
Grade  A,  71.1°  C.  (160°  F.).  . 
Grade  B,  62.8°  C.  (145°  F.).  . 
Grade  C,  62.8°  C.  (145°  F.).. 

(Infusion  agar 

1.27 
4.26 
1.71 
2.34 
6.98 
6.50 
4.55 
13.09 

0.37 
.31 
.12 
.39 
1.48 
2.06 
1.59 
4.49 

0.05 
1.55 
.10 
0 
1.2T 
2.05 
.19 
1.13 

0.05 
.08 
.01 
.03 
1.86 
.51 
1.24 
1.25 

\Casein  agar 

(Infusion  agar     .... 

\Casein  agar  

\Casein  agar 

/Infusion  agar 

\Casein  agar 

THE  EFFECT  OF  PASTEURIZATION  ON  THE  NUMBER,  OF  BACTERIA 
OF   DIFFERENT   GROUPS   IN  MILK. 

In  order  to  show  the  effect  of  pasteurization  on  the  numbers  of 
the  bacteria  of  different  groups,  Table  16  has  been  arranged.  This 
shows  the  calculated  number  of  bacteria  in  each  group  determined 
from  the  average  total  counts  on  infusion  agar  and  the  group  per- 
centages as  shown  by  the  milk-tube  method.  To  illustrate  this  fur- 
ther take  the  samples  from  dairy  A,  as  shown  in  Table  16.  The 
bacterial  count  of  12  samples  of  raw  milk  averaged  was  21,100. 
The  averaged  group  percentages  of  the  12  samples  was  33.85  per 
cent  of  the  acid-coagulating  group,  6.85  per  cent  of  the  acid  group, 
and  so  on.  The  acid-coagulating  group  contained  on  an  average 
33.85  per  cent  of  the  average  total  count,  or  approximately  7,160 
bacteria  per  cubic  centimeter.  The  other  bacterial  numbers  were 
calculated  in  the  same  way. 


36 


BACTERIA   WHICH    SURVIVE   PASTEURIZATION. 


TABLE  16. — Comparison  of  the  number  of  bacteria  of  different  groups  in  milk  before  and 

after  pasteurization. 


Average  total  count  per  cubic  centimeter. 

Average 
group 
per- 
centage. 

Group. 

Calculated 
number  of 
each  group 
per  cubic 
centimeter. 

33.85 

Acid-coagulating        

7,160- 

6.85 

Acid  

1,450 

Raw  milk,  21,  100.    Average  of  12  samples.    Dairy  A. 

43.13 

Inert  

9,120 

3.33 

Alkali  

705 

12.81 

Peptonizing 

2,700 

11.85 

Acid-coagulating  

50 

61.25 

Acid  

257 

Pasteurized  mflk,  62.8°  C.  (145°  F.),  30  minutes,  420. 
Average  of  11  samples     Dairy  A 

22.06 

Inert  

92 

.36 
4.47 

Alkali  
Peptonizing 

2 
19 

12.98 

Acid-coagulating  

368,800 

9.74 

Acid  

276,700 

Raw   mflk,   2,840,000.    Average   of   17     samples. 
Dairies  B  and  C 

43.51 

Inert  

1,236,000 

19.66 
14  10 

Alkali  

Peptonizing 

558,000 
400,000 

31.89 

Acid-coagulating      

3,760 

34.87 

Acid  

4,110 

24,00 

Inert  

2.830 

5.63 

Alkali     

660 

3.59 

Peptonizing  

420 

36.17 

Acid-coagulating         

8,200,000 

10.71 

Acid  

2,430,000 

Dairy  D  

29.31 

Inert  

6,  700,  000 

6.47 
17.31 

Alkali  
Peptonizing  

1,  480,  000 
3,900,000 

17  91 

Acid-coagulating 

14.900 

61.87 

Acid          

51,500 

9.06 

Inert  

7.500 

9.77 

Alkali  

8,130 

1.39 

Peptonizing  

1,150 

The  most  striking  features  of  the  table  is  the  high  number  of  acid- 
forming  bacteria  in  proportion  to  the  low  number  of  peptonizers  in 
the  pasteurized  milk  and  the  marked  difference  in  the  numbers  of 
peptonizing  bacteria  before  and  after  pasteurization.  The  milk  from 
dairy  A  was  exceptionally  good,  the  average  total  count  of  the  raw 
milk  being  21,100  and  after  pasteurization  for  30  minutes  at 
62.8°  C.  (145°  F.)  only  420  per  cubic  centimeter.  The  peptonizers 
were  reduced  from  2,700  to  19  per  cubic  centimeter. 

The  milk  from  dairies  B  and  C  was  of  poorer  quality,  the  average 
count  of  the  raw  milk  being  2,840,000.  After  pasteurization  the 
count  was  1 1 ,700.  The  peptonizers  were  reduced  by  the  process  from 
400,000  to  420  per  cubic  centimeter.  In  the  last  part  of  the  table  the 
results  of  the  examination  of  poor  milk  is  shown.  The  average  count 
was  22,800,000  bacteria  per  cubic  centimeter  before  and  83,000  after 
pasteurization  for  30  minutes  at  62.8°  C.  (145°  F.).  The  raw  milk 
contained  an  average  of  3,900,000  peptonizers  per  cubic  centimeter 
and  after  pasteurization  only  1,150  per  cubic  centimeter.  Through- 
out the  whole  series  may  be  noticed  the  low  number  of  alk ah' -forming 
bacteria  in  the  pasteurized  milk  in  proportion  to  those  in  the  raw  milk. 
After  a  comparison  is  made  of  the  kinds  of  bacteria  in  the  raw  milk 


BACTEKIAL  DEVELOPMENT  IN  MILK.  37 

from  dairies  B  and  C  and  in  the  extremely  poor  milk  from  dairy  D  with 
the  same  milk  after  pasteurization  there  should  be  no  question  as  to 
the  beneficial  effect  of  the  process. 

THE  BACTERIAL  DEVELOPMENT  IN  MILK  PASTEURIZED  IN  THE 
LABORATORY  AND  HELD  AT  DIFFERENT  TEMPERATURES. 

After  having  determined  the  groups  of  bacteria  which  survived 
pasteurization,  their  subsequent  development  was  traced  in  a  few 
samples  of  milk. 

The  changes  in  the  bacterial  flora  of  milk  pasteurized  for  30  minutes 
at  62.8°  C.  (145°  F.)  during  storage  at  different  temperatures  has  been 
studied  in  a  poor,  medium,  and  good  grade  of  milk.  One  sample  of 
each  grade  of  milk  was  divided  and  each  part  pasteurized  in  a  sterile 
flask.  After  pasteurization  each  sample  was  held  in  the  ice  box  at 
approximately  7.23°  C.  (45°  F.)  for  18  hours;  then  one  flask  was  held 
at  room  temperature,  the  other  was  kept  in  the  ice  box.  After  pas- 
teurization and  on  each  successive  day  each  sample  was  plated  to 
determine  the  number  of  bacteria  which  were  then  differentiated  by 
the  milk-tube  method.  On  account  of  the  great  amount  of  work 
involved  and  time  consumed  in  determining  the  bacterial  flora  by  the 
milk-tube  method  only  one  sample  of  each  grade  was  studied. 


38 


BACTERIA  WHICH   SURVIVE   PASTEURIZATION. 


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BACTERIAL  DEVELOPMENT  IN  MILK. 


39 


The  daily  bacterial  group  percentages  are  shown  in  Table  17.  The 
room  temperature  varied  from  21.1°-23.9°  C.  (70°-75°F.)  while  the 
temperature  of  the  ice  box  ranged  from  8 °-10°C.  (46.4°-50°F.).  The 
bacterial  group  percentages  in  each  raw  milk  are  shown  followed  by 
the  "0"  day  determination  which  gives  the  group  percentages  imme- 
diately after  pasteurization.  Sample  G  represented  a  poor  quality  of 
milk,  H  a  milk  of  medium  quality,  such  as  is  usually  commercially 
pasteurized,  and  sample  K  an  excellent  grade  of  milk  containing  only 
a  few  thousand  bacteria  per  cubic  centimeter.  The  samples  of  milk 


FIG.  17. — Daily  changes  in  the  bacterial  group  relations  in  pasteurized 
milk.    Sample  G  held  in  the  ice  box. 


FIG.  16. — Daily  changes  in 
the  bacterial  group  rela- 
tions in  pasteurized  milk. 
Sample  G  held  at  room 
temperature. 

were  pasteurized  and  held  so  as  to  prevent  any  possible  reinfection. 
In  order  to  show  the  changes  in  the  groups  more  plainly,  the  results 
from  Table  17  have  been  plotted.  Figure  16  shows  the  changes  in 
the  bacterial  flora  of  sample  G  held  at  room  temperature.  This  sam- 
ple showed  a  rennet  curd  on  the  third  day.  While  there  was  a  little 
acid  development  during  the  first  three  days,  a  rapid  increase 
occurred  from  the  third  to  the  fourth  day.  From  the  figure  it  may  be 
seen  that  there  was  a  rapid  increase  in  the  percentage  of  the  pepto- 
nizing  group  from  4.75  per  cent  on  "0"  day  to  20  per  cent  after  one 


40 


BACTERIA   WHICH    SURVIVE   PASTEURIZATION. 


day,  then  to  80.95  per  cent  after  two  days,  followed  by  a  drop  to  20.79 
per  cent  the  third  day.  Corresponding  to  the  increase  in  the  propor- 
tion of  the  peptonizing  group  there  was  a  decrease  in  the  acid  group 
followed  by  an  increase  on  the  third  day.  The  acid  group  comprised 
85.71  per  cent  of  the  bacteria  at  first,  then  dropped  to  67.69  per  cent 
after  one  day,  then  to  9.52  on  the  second  day,  then  increased  53.96 
per  cent.  The  percentage  of  the  alkali  group  throughout  was  low. 
As  the  plot  shows,  there  was  a  rapid  increase  hi  the  peptonizing  bac- 
teria which  produced  a  rennet  curd  on  the  third  day.  Up  to  that 

time  the  development  of  the  acid-forming 
bacteria  was  slow,  and  while  there  was 
some  development  of  acid,  apparently  not 
enough  was  formed  to  check  the  develop- 
ment of  the  peptonizing  group.  To  sum- 
marize, it  may  be  said  that  in  sample  G, 
held  at  room  temperature,  there  was  a 
rapid  growth  of  peptonizing  organisms 
which  produced  a  rennet  curd,  followed  by 
acid  development  produced  by  the  growth 
of  the  acid-forming  bacteria. 

The  other  portion  of  sample  G  was  held 
in  the  ice  box.  Figure  17  shows  the  re- 
sults graphically.  It  may  be  seen  that 
there  was  only  a  slight  acid  development 
during  the  13  days'  holding  period.  The 
most  striking  point  is  the  restriction  of  the 
development  of  the  peptonizing  group  by 
the  low  temperature  at  which  the  sample 
was  held.  A  comparison  of  the  plot  of  the 
peptonizing  group  in  figures  16  and  17 
shows  this  point  plainly.  There  was  a 
slow  bacterial  development  during  the  first 
six  days  as  shown  by  the  total  counts. 
During  that  time  the  percentage  of  the  acid 
group  was  high.  From  the  seventh  to  the 


2       3 
US    J8 


\\ 

r 


FIG.  18. — Dailychangesinthe  bacterial     ~.    ,,- 

group  relations  m  pasteurized  milk,  ninth  day  the  per  cent  of  the  acid  group 
sample  H  held  at  room  temperature,  was  low,  with  a  corresponding  increase  in 
the  alkali  and  inert  groups.  From  the  ninth  day  the  percentage  of 
the  acid  group  began  to  increase  and  the  acidity  also  increased. 
The  bacterial  groups  were  not  studied  after  the  thirteenth  day,  but 
the  sample  was  held  and  acidity  was  determined  daily.  It  was 
found  that  the  milk  eventually  soured.  After  eight  days  an  old 
taste  developed  which  would  prevent  its  consumption  as  a  food. 

The  changes  in  the  bacterial  flora  of  a  better  grade  of  milk  repre- 
sented by  sample  H  were  much  different  when  the  milk  was  held  at 
room  temperature.  As  may  be  seen  from  figure  18  the  milk  soured 
with  a  constantly  increasing  percentage  of  the  acid  group  and  decrease 


BACTERIAL  DEVELOPMENT  IN  MILK. 


41 


in  the  other  group  percentages.  The  percentage  of  the  acid  group 
increased  from  58.69  after  pasteurization  to  91.71  after  six  days. 
/oo 


s 

Flo.  19.— Daily  changes  in  the  bacterial  group  relations  in  pasteurized  milk.    Sample  II  held  in  ice  box. 

The  percentage  of  the  peptonizing  group  was  0.54  immediately  after 

heating  and  none  appeared  in  the  plates  in  the  later  examinations. 

The  changes  in  the  groups  of  the  other  portion  of  sample  II  held  in 

the  ice  box  are  shown  in  figure  19.     The  development  of  the  alkali 


42 


BACTERIA  WHICH   SURVIVE   PASTEURIZATION. 


group  after  four  days  is  plainly  shown.  With  the  increase  in  the  per- 
centage of  the  alkali  group  there  was  a  corresponding  decrease  in  the 
acid  group.  After  10  days  the  percentage  of  the  alkali  group  began 
to  decrease  and  the  acid  group  to  increase.  The  percentage  of  the 
peptonizing  group  was  low  through  the  whole  period.  The  acidity 
did  not  change  until  the  twelfth  day.  After  11  days  the  milk  had  an 
old  taste  and  would  not  be  used. 

The  changes  in  the  bacterial  groups  in  the  best  grade  of  milk  repre- 
sented by  sample  K  were  different  from  the  others  when  the  milk  was 


FIG.  20.—  Daily  changes  in 
the  bacterial  group  rela- 
tions in  pasteurized  milk. 
Sample  K  held  at  room 
temperature. 


FIG.  21.  —  Daily  changes  in  the  bacterial  group  relations  in  pasteurized 
milk.    Sample  K  held  in  the  ice  box. 


held  at  room  temperature.  After  six  days  the  milk  was  putrid  and 
digested,  but  had  not  coagulated.  As  may  be  seen  from  figure  20, 
there  was  no  acid  formed.  On  the  second  day  the  peptonizing  bac- 
teria began  to  increase  rapidly,  and  on  the  fourth  day  only  organisms 
of  the  peptonizing  group  were  found.  The  acid  group  gradually 
decreased  until  the  second  day,  after  which  none  appeared  on  the 
plates.  The  extreme  changes  in  the  group  percentages  were  due  to 
the  low  number  of  bacteria  in  the  milk.  When  only  a  few  colonies 
appeared  on  a  plate  the  determination  of  the  groups  was  of  course, 


QUALITATIVE    STUDY    OF    GROUPS.  43 

much  less  accurate  than  when  100  or  more  colonies  are  studied  by  the 
milk-tube  method. 

The  restraining  effect  of  low  temperatures  on  the  development  of 
the  peptonizing  group  is  again  plainly  shown  in  figure  21,  which 
represents  the  bacterial  group  changes  in  the  other  portion  of  sample 
K  held  in  the  ice  box.  No  peptonizing  bacteria  were  found  on  the 
plates  throughout  the  whole  series  and  none  of  the  alkali  group 
appeared  until  the  twelfth  day.  The  acid  group  predominated 
throughout  with  considerable  fluctuation  due  to  inaccuracy  on  account 
of  low  bacterial  numbers.  The  total  counts  were  very  low  until  the 
eleventh  day,  after  which  there  was  a  rapid  increase.  After  17  days 
an  old  taste  was  noticed  and  at  that  time  the  acidity  began  to  increase 
slowly. 

These  results  are  shown  merely  to  indicate  the  possible  changes 
which  may  take  place  in  milk  pasteurized  in  sealed  bottles.  It  is 
clear  that  there  is  a  delicate  balance  between  the  various  bacterial 
groups  which  may  be  influenced  by  conditions  of  temperature  and 
time  so  that  various  effects  may  be  produced.  These  results  can  not 
be  applied  to  commercial  problems,  since  in  laboratory  experiments 
it  is  impossible  to  duplicate  commercial  conditions.  In  order  to 
determine  the  changes  in  milk  commercially  pasteurized  in  sealed 
bottles  a  study  must  be  carried  on  only  under  commercial  conditions. 

QUALITATIVE     STTTDY    OF    THE     GROUPS    OF    BACTERIA    WHICH 
SURVIVE    PASTEURIZATION. 

During  the  determination  of  the  percentage  of  the  bacterial  groups 
cultures  were  selected  for  further  study.  These  were  selected  from 
milk  pasteurized  at  62.8°  C.  (145°  F.)  for  30  minutes.  The  cultures 
were  purified  by  plating,  then  examined  microscopically  and  for  their 
cultural  reactions.  In  the  tables  of  the  Appendix  the  complete  reac- 
tions are  shown. 

THE    ACID-FORMING    GROUP   OF   BACTERIA. 

The  acid-forming  group  of  bacteria  have  been  grouped  arbitrarily 
as  shown  in  figure  22.  One  hundred  and  thirty-two  cultures  were 
examined  and  were  divided  into  four  classes.  The  first  class  comprised 
89.39  per  cent  of  the  cultures.  The  reactions  of  this  class  are  shown  in 
figure  22.  All  the  organisms  were  cocci  which  produced  no  liquefac- 
tion of  gelatin.  The  Gram  stain  was  either  positive  or  negative, 
nitrate  was  not  reduced,  and  some  formed  pigment  while  others  did 
not.  Dextrose,  galactose,  and  lactose  were  fermented.  Some  cul- 
tures fermented  saccharose.  Of  those  that  fermented  saccharose,  the 
ability  to  ferment  raffinose,  starch,  inulin,  mannite,  glycerin,  and 
salicin  varied  as  may  be  seen.  The  same  may  be  said  regarding  those 
cultures  which  did  not  ferment  saccharose.  The  second  class  was  made 


44 


BACTERIA   WHICH   SURVIVE   PASTEURIZATION. 


up  of  nonliquefying  bacilli,  Gram  positive,  nitrate  reduction  positive  or 
negative,  and  all  produced  pigment.  Dextrose,  galactose,  lactose, 
and  saccharose  were  fermented;  raffinose  positive  or  negative,  starch 
positive  or  negative,  inulin  not  fermented,  mannite  positive  or  nega- 
tive, glycerin  not  fermented,  and  salicin  positive  or  negative.  This 
class  comprised  3.03  per  cent  of  the  acid  group  cultures.  The  third 


TOT4L  MUMfffft  /32. 


CLASS  /V. 


FIG.  22.— The  acid-forming  group  of  bacteria. 

class  contained  6.81  per  cent  of  the  cultures  and  was  made  up  of 
liquefying  cocci,  Gram  positive,  nitrate  reduction  positive  or  negative, 
and  none  produced  pigment.  The  acid  production  was  as  follows: 
Dextrose,  galactose,  lactose,  and  saccharose  positive ;  raffinose,  inulin, 
and  starch  negative ;  mannite  and  glycerin  positive  or  negative ;  and 
salicin  negative. 

One  culture  formed  the  fourth  class.     It  was  a  liquefying  Gram 
positive  bacillus  which  reduced  nitrates  and  formed  no  pigment. 


QUALITATIVE    STUDY    OF    GROUPS. 


45 


Acid  was  formed  in  dextrose,  lactose,  saccharose,  and  glycerin.  The 
complete  reactions  of  each  variety  of  bacteria  are  shown  in  Table  I 
of  the  Appendix. 

In  general  it  may  be  said  that  the  majority  of  the  acid  group  which 
survived  pasteurization  at  62.8°  C.  (145°  F.)  were  nonliquefying 
cocci  which  varied  widely  in  their  ability  to  ferment  the  various 
sugars.  A  small  percentage  of  acid-forming  bacilli  also  survived, 
together  with  a  few  acid-liquefying  bacteria. 

THE    INERT    GROUP    OF    BACTERIA. 

The  study  of  the  inert  group  has  not  been  given  much  attention. 
All  bacteria  which  produced  no  change  in  litmus  milk  after  14  days' 
incubation  at  30°  C. 
(86°  F.)  were  classed 
according  to  the  milk- 
tube  method  as  inert 
organisms.  The  group 
would  therefore  contain 
not  only  those  bacteria 
which  did  not  produce 
any  change  in  milk  un- 
der any  condition,  but 
also  any  organisms 
which  did  not  happen 
to  grow  in  the  milk 
tubes  and  those  which 
might  produce  a  change 
in  milk  after  the  14 
days'  incubation 
period. 

In  a  number  of  sam- 
ples agar  streak  cul- 
tures were  made  from 
the  litmus  milk  tubes  which  showed  no  change.  Sometimes  none  of  the 
agar  cultures  showed  growth;  in  other  cases  approximately  2  percent,  5 
per  cent,  25  per  cent,  80  per  cent,  and  occasionally  100  per  cent  showed 
growth  on  agar  streaks.  When  those  agar  cultures  were  reinoculated 
into  litmus  milk,  some  would  not  produce  any  reaction  and  others  a 
slight  alkaline  reaction  after  long  incubation.  Often  a  yellow  color 
was  noticed  in  the  litmus  milk  culture  which  indicated  an  inert  yellow 
pigment-forming  organism. 

THE    ALKALI-FORMING   GROUP   OF   BACTERIA. 

The  alkali-forming  bacteria  constitute  a  group  which  has  been  given 
but  little  study  in  connection  with  the  bacterial  flora  of  milk.  In 


FIG.  23. — The  alkali-forming  group  of  bacteria. 


46 


BACTEEIA   WHICH    SURVIVE   PASTEURIZATION. 


the  differentiation  of  the  various  groups  on  litmus  lactose  gelatin 
plates,  organisms  of  this  group  would  be  entirely  missed,  but  they 
are  easily  found  by  the  milk-tube  method.  Forty-three  of  these 
organisms  isolated  from  pasteurized  milk  have  been  studied  and 
grouped  arbitrarily,  as  shown  in  figure  23.  The  alkali  group  was 
made  up  of  72.09  per  cent  of  cocci  and  27.90  per  cent  of  bacilli. 


FIG.  24. — The  peptonizing  group  of  bacteria. 

It  is  not  necessary  to  go  into  minute  details  regarding  these  organisms, 
for  their  characteristics  are  plainly  shown  in  the  figure.  They  did 
not  liquefy  gelatin,  and  produced  a  strong  alkaline  reaction  in  litmus 
milk.  No  acid  was  produced  in  the  sugars,  starches,  alcohols,  or  in 
the  glucosid  used  in  the  investigation,  but  an  alkaline  reaction  was 
often  found.  In  litmus  milk  often  enough  alkali  was  produced  to 


QUALITATIVE   STUDY   OF   GROUPS.  47 

dissolve  the  casein.  That  will  always  happen  if  the  period  of  inctf- 
bation  is  long  enough.  The  complete  characteristics  are  shown  in 
the  Appendix  in  Table  II.  A  more  extended  study  of  the  alkali  group 
is  under  way  in  the  research  laboratories  of  this  division. 

THE    PEPTONIZING    GROUP   OF    BACTERIA. 

The  peptonizing  bacteria  include  only  those  which  peptonize  the 
casern  of  milk.  Fifty  of  this  type  of  organisms  were  studied,  and 
their  characteristics  are  shown  fuUy  in  the  Appendix  in  Table  III. 
They  were  divided  arbitrarily  into  four  classes,  as  shown  in  figure  24. 
It  may  be  seen  that  the  bacteria  of  classes  1  and  2  did  not  liquefy 
gelatin  during  30  days'  incubation,  but  did  peptonize  casein.  The 
peptonization  of  casein  was  determined  by  streak  cultures  on  casein 
agar  slopes  by  the  following  method : 

After  14  days'  incubation  the  slopes  were  flowed  with  N/lOth  lactic 
acid.  If  no  peptonization  has  taken  place  the  casein  will  be  precipi- 
tated by  the  acid.  If  peptonization  has  gone  on,  then  the  casein  agar 
will  remain  clear,  showing  that  the  casein  has  been  dissolved.  The 
use  of  casein  agar  for  the  determination  of  peptonizing  bacteria  in 
milk  is  fully  described  in  another  publication  1  of  this  department. 
It  will  be  seen  from  figure  24  that  classes  1  and  2  comprise  both  cocci 
and  bacilli,  some  of  which  did  not  ferment  sugars,  while  others  pro- 
duced acid.  Classes  3  and  4  is  made  up  of  bacteria  which  liquefy 
gelatin  and  may  or  may  not  ferment  the  sugars.  It  is  interesting  to 
note  that  out  of  225  cultures  of  bacteria  studied  only  3,  or  1.35  per 
cent,  formed  spores.  Those  3  cultures  were  found  among  the  50 
peptonizing  organisms  making  only  6  per  cent  of  the  peptonizing 
spore-forming  organisms. 

THE    GAS-FORMING    BACTERIA. 

During  the  study  of  225  cultures  selected  at  random  from  the 
samples  of  pasteurized  milk  only  two  organisms  were  found  which 
produced  gas  in  milk.  One  of  the  gas-forming  bacteria,  culture  417, 
was  a  Gram  negative  short  bacillus  which  peptonized  casein  but  did 
not  liquefy  gelatin.  It  produced  gas  in  lactose  broth  and  acid  in 
dextrose,  saccharose,  raffinose,  starch,  and  salicin  broths.  No  acid 
was  formed  in  lactose,  galactose,  mannite,  glycerin,  or  inulin  broths. 
Nitrate  solution  was  reduced.  Gas  was  produced  in  litmus  milk 
and  the  milk  was  curdled  with  some  peptonization.  It  is  evident 
that  this  bacillus  did  not  belong  to  the  colon-aerogencs  group. 

The  other  organism  is  an  entirely  new  type  so  far  as  known.  This 
culture  known  as  Z  produced  gas  in  milk  but  not  in  lactose  broth. 

i  Ayers,  8.  Henry.    Casein  media  adapted  to  determining  bacteria  in  milk.    United  States  Department 
ol  Agriculture,  Bureau  of  Animal  Industry,  Twenty-eighth  Annual  Report,  1911  (in  press). 


48 


BACTERIA    WHICH    SURVIVE    PASTEURIZATION. 


Culture  Z  was  a  long  motile  Gram  negative  bacillus.  In  a  hanging 
drop  preparation  a  few  cells  had  an  enlargement  at  one  end  which 
probably  represented  spores,  although  it  was  impossible  to  demon- 
strate their  presence  by  staining  reactions.  Their  survival  of  an 
exposure  to  a  temperature  of  93.3°  C.  (200°  F.)  for  30  minutes  seemed 
to  affirm  the  presence  of  spores.  It  grew  slowly  on  agar  streaks  and 
produced  a  slimy  growth.  A  2  days'  old  culture  often  showed 
extremely  long  types  of  organisms.  Acid  was  produced  in  dextrose, 
galactose,  and  salicin  broths,  but  not  in  saccharose,  mannite,  gly- 
cerin, raffinose,  inulin,  or  starch  broths.  When  these  broths  were 
used  for  gas  production  it  was  found  in  one  experiment  that  culture 
Z  produced  gas  in  raffinose,  mannite,  and  starch 
broths  after  14  days'  incubation  at  30°  C.  (86°  F.), 
but  none  in  any  of  the  other  broths.  This  ex- 
periment was  repeated  and  no  gas  was  found  in 
any  of  the  broths.  Nitrate  solution  was  reduced 
and  gelatin  was  not  liquefied  after  30  days'  incuba- 
tion at  18°  C.  (64.4°  F.).  This  culture  produced 
acid  and  gas  in  milk  and  coagulated  it  after  14 
days.  The  ability  to  produce  gas  in  milk  through 
a  long  period  is  one  of  the  striking  characteristics 
of  this  organism.  In  order  to  determine  how 
long  culture  Z  would  continue  to  produce  gas  in 
milk,  a  25  cubic  centimeter  burette  was  inverted 
and  inserted  through  a  rubber  cork  into  an  Erlen- 
meyer  flask  containing  200  cubic  centimeters  of 
milk,  as  shown  in  figure  25.  By  opening  the  stop- 
cock of  the  burette  the  milk  could  be  drawn  to 
the  top  and  the  tube  filled.  After  sterilizing,  the 
milk  was  inoculated  with  culture  Z  and  incubated 

at  30°  C.  (86°  F.).  The  gas  produced  Was  re- 
determination  of  gas  pro-  corded  daily  and  when  the  burette  was  filled 
with  gas  the  stopcock  was  opened  and  the 
tube  was  again  filled  with  milk.  In  that  way  it  was  possible 
to  record  gas  production  from  milk  over  an  indefinite  period. 
As  may  be  seen  from  figure  26,  this  organism  was  able  to  pro- 
duce gas  through  a  period  of  46  days.  The  fermentation  began 
after  24  hours'  incubation  when  from  3  to  4  cubic  centimeters 
of  gas  was  formed  daily  until  the  fourteenth  day,  when  the  milk 
was  curdled.  At  that  time  the  daily  gas  production  increased  and 
was  sometimes  as  high  as  15  cubic  centimeters.  The  increased 
gas  production  continued  until  the  thirty-first  day,  when  it  was 
reduced  to  approximately  4  cubic  centimeters  per  day.  The  gas 
produced  was  hydrogen  and  carbon  dioxide.  The  ability  of  culture 


TUBS 


FIG.  25.— Apparatus  for  the 


QUALITATIVE   STUDY   OF   GROUPS. 


49 


Z  to  produce  gas  in  milk  continuously  for  a  long  period,  together 
with  the  fact  that  while  gas  was  formed  in  milk  lactose  in  broth  was 
not  fermented,  differentiates  this  organism  from  the  ordinary  types 
of  gas  formers.  A  more  extended  study  of  this  type  of  organism  is 
under  way  in  the  research  laboratories  of  this  division. 

The  survival  of  pasteurization  by  gas-forming  bacteria  is  of  con- 
siderable importance,  since  it  has  been  suggested  to  make  the  pres- 
ence of  the  colon  group  an  indication  of  recontamination  of  pasteur- 


eed 

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Flo.  26.— Gas  produced  by  culture  / . 

ized  milk  or  of  inefficient  pasteurization.  Several  boards  of  health 
are  at  present  using  such  a  test.  If  gas-forming  organisms  do  not 
survive  pasteurization,  then  of  course  their  presence  in  pasteurized 
milk  means  recontamination  or  inefficient  pasteurization.  If,  how- 
ever, certain  types  of  gas-forming  bacteria  do  survive  the  process, 
then  the  test  is  of  no  value.  In  order  to  determine  if  gas-forming 
bacteria  survived  pasteurization  for  30  minutes  at  62.8°  C.  (145°  F.), 
19  samples  of  raw  milk  were  heated  in  sterile  flasks.  Fermentation 


50 


BACTERIA   WHICH    SURVIVE    PASTEURIZATION. 


tubes  in  sets  of  four  were  then  inoculated  with  1  cubic  centimeter  and 
5  cubic  centimeters  of  both  the  raw  and  the  pasteurized  milk.  The 
results  are  shown  in  Table  18.  Twelve  of  the  samples  of  raw  milk 
showed  the  presence  of  gas-forming  organisms  in  lactose  bile  tubes 
when  1  cubic  centimeter  of  milk  was  used  for  inoculation.  After 
pasteurization  three  samples  showed  gas  when  1  cubic  centimeter 
was  used  and  eight  samples  when  5  cubic  centimeters  of  milk  was 
used.  When  dextrose  liver  broth  fermentation  tubes  were  used,  a 
larger  number  of  samples  of  the  pasteurized  milk  showed  the  pres- 
ence of  gas-forming  bacteria.  It  may  be  seen  from  the  table  that 
the  number  of  gas  producers  in  the  raw  and  pasteurized  milk  was 
low  as  determined  by  the  dilution  and  the  number  of  tubes  of  each 
set  of  four  which  showed  gas.  For  example,  sample  1,  four  tubes  of 
lactose  bile,  each  inoculated  with  1  cubic  centimeter  of  raw  milk, 
showed  no  gas,  which  means  that  no  gas-forming  organisms  were 
present  in  4  cubic  centimeters  of  the  raw  milk.  Four  tubes,  each 
inoculated  with  5  cubic  centimeters  of  the  same  milk  pasteurized, 
showed  gas  in  two  tubes.  That  means  at  least  one  gas-forming 
organism  in  every  10  cubic  centimeters  of  the  milk. 

TABLE  18. — Gas  production  in  lactose-bile  and  dextrose-liver  broths  inoculated  with  raw 
milk  and  milk  pasteurized  at  66.8°  C.  (145°  F.)  for  30  minutes.  Four  tubes  used  in 
each  dilution. 


Gas  produced  in— 

Lactose-bile  broth. 

Dextrose-liver  broth. 

Sample 
num- 

Inocu- 

Inocu- 

Inocu- 

Inocu- 

ber. 

Inocu- 

lated 

lated 

Inocu- 

lated 

lated 

lated 

withl 

with  5 

lated 

withl 

with  5 

withl 

c.  c.  pas- 

c. c.  pas- 

with 5 

c.  c.  pas- 

c. c.  pas- 

c. c.  raw 

teur- 

teur- 

c. c.  raw 

teur- 

teur- 

milk. 

ized 

ized 

milk. 

ized 

ized 

milk. 

milk. 

milk. 

milk. 

Tubes. 

Tubes. 

Tubes. 

Tubes. 

Tubes. 

Tubes. 

1 

0 

0 

2 

0 

3 

2 

2 

0 

0 

0 

0 

3 

2 

3 

0 

0 

0 

0 

1 

0 

4 

3 

0 

1 

1 

0 

1 

5 

0 

0 

0 

3 

0 

2 

6 

3 

0 

3 

3 

0 

2 

7 

3 

0 

1 

3 

0 

1 

8 

0 

0 

0 

0 

0 

2 

9 

2 

0 

0 

0 

0 

2 

10 

2 

0 

0 

0 

0 

0 

11 

1 

0 

0 

0 

3 

2 

1  12 

0 

0 

0 

0 

13 

4 

0 

0 

3 

1 

4 

14 

3 

0 

0 

4 

0 

0 

15 

1 

3 

1 

2 

1 

1 

16 

4 

2 

4 

4 

2 

2 

17 

4 

0 

0 

4 

0 

2 

18 

4 

19 

3 

1 

4 

1  Milk  incubated  at  37°  C.  for  18  hours  and  then  pasteurized. 

Litmus  lactose  agar  plates  were  made  and  inoculated  from  almost 
every  sample  which  showed  gas  in  the  bile  tubes,  but  no  organisms 
of  the  colon  type  were  found.  The  only  gas  producer  which  was 


THEEMAL   DEATH    POINT.  51 

found  was  a  spore-forming  anaerobic  bacillus  of  the  butyric  group. 
One  of  these  organisms,  known  as  culture  T,  produced  acid  in  dex- 
trose, galactose,  saccharose,  raffinose,  and  starch  broths,  and  gas  in 
dextrose,  galactose,  lactose,  saccharose,  and  starch  broths.  In  this 
work  no  organisms  of  the  colon  group  were  found  to  survive  pas- 
teurization, but  gas  was  often  produced  in  lactose  bile  tubes  inocu- 
lated with  pasteurized  milk.  The  gas  production,  however,  was  not 
due  to  organisms  of  the  colon  group,  but  to  those  of  the  butyric-acid 
group.  This  anaerobic  butyric  bacillus  produced  gas  in  lactose  bile 
tubes  and  would  appear  in  the  preliminary  tests  for  the  colon  bacillus. 
Therefore,  if  only  lactose  bile  fermentations  were  used  to  determine 
the  presence  of  the  colon  group  in  pasteurized  milk,  the  anaerobic 
butyric  types  of  bacteria  would  produce  fermentations  which  would 
be  taken  as  an  indication  of  the  colon  group.  It  is  evident,  then,  that 
the  presence  of  the  colon  group  of  organisms  in  milk  must  be  deter- 
mined by  something  more  than  simply  lactose  bile  fermentation 
tubes.  These  results  are  by  no  means  conclusive,  for  if  a  larger 
number  of  samples  had  been  examined  colon  bacilli  might  have  been 
found  which  survived  pasteurization.  De  Jong  and  De  Graaff,1 
working  on  various  strains  of  Bacillus  coli,  found  some  forms  which 
required  30  minutes  heating  at  70°-72°  C.  (158°-161.6°  F.)  to 
destroy.  Zelenski  2  also  found  some  strains  of  B.  coli  which  resisted 
high  temperatures.  Evidently  resistant  types  of  the  colon  group  do 
exist  and  that  fact  complicates  the  value  of  the  colon  test  for  reinfec- 
tion of  pasteurized  milk. 

THERMAL    DEATH    POINTS    OF    BACTERIA    WHICH    SURVIVE 
PASTEURIZATION. 

As  a  matter  of  purely  scientific  interest  the  thermal  death  points 
of  several  bacteria  which  survived  pasteurization  were  determined. 
The  determinations  were  made  by  heating  2  days'  old  lactose  dibasic 
broth  cultures  for  30  minutes  in  Sternberg  bulbs.  After  heating,  the 
contents  of  the  bulbs  were  inoculated  into  litmus  milk  tubes.  Cul- 
tures were  selected  from  the  acid,  alkali,  and  peptonizing  groups  for 
the  experiment.  The  results  are  shown  in  Table  19.  Culture  40  of 
the  acid  group  stood  as  high  as  79.4°  C.  (175°  F.).  Numbers  58  and 
156  were  destroyed  at  73.9°  C.  (165°  F.)  and  culture  81  at  65.6°  C. 
(150°  F.).  One  culture  of  the  alkali  group  was  destroyed  at  65.6°  C. 
(150°  F.).  Culture  450  of  the  peptonizing  group  was  destroyed  at 
71.1°  C.  (160°  F.),  while  the  thermal  death  point  of  culture  456  was 
indefinite,  although  about  79.4°  C.  (175°  F.). 

i  De  Jong,  D.  A.,  and  De  Graaff,  W.  C.  Die  Coll-Kontrolle  der  pasteurisierten  Milch.  [Nederlandsch 
Weekblad  voor  Zuivell>ereiding  en  Veeteelt,  vol.  12,  Nos.  37  and  38,  1906].  Abstract:  Milchwirtschaft- 
liches  Zentralblatt,  vol.  3,  No.  (i,  pp.  265-2<i8.  Leipzig,  June,  1907. 

*  Zelenski,  Thaddeus.  Zur  Frage  der  Pasteurisation  der  Siiuglingsmilch.  Jahrbuch  fur  Kiiiderheil- 
kunde,  vol.  63,  pp.  288-307.  Berlin,  1900.  Abstract:  Centralblatt  fiir  Bakteriologie,  Parasitenkunde  und. 
Infektionskrankheiten.  Abteilung  2,  vol.  18,  No.  4-C,  p.  175.  Jena,  Mar.  14, 1907. 


52  BACTERIA   WHICH   SURVIVE   PASTEURIZATION. 

TABLE  19. —  Thermal  death  point  of  bacteria  surviving  pasteurization. 


Culture. 

30  minutes  in  Sternberg  bulbs  at  — 

^ 

ft 

CO 

d 

IN 

r~ 

fe 
§ 
d 

s 

fc 

>o 
•^ 

ptj 

s 

FH 
S 

fe 

8 

h 
8 

fe 
S 

SH 

S 

d 

d 

d 

d 

d 

d 

d 

00 

s 

o 

8 

1 

»~ 

Ol 
M 

i- 
52 

•* 

gi 

ACID  GROUP. 

Culture  58 

++ 
++ 
++ 

++ 

++ 

++ 
++ 

++ 
++ 

++ 

++ 
++ 

Culture40  

+  + 

+  + 

H  — 

Culture  81 

Culture  15C 

+  + 

++ 

++ 

ALKALI  GROUP. 

Culture  311 

+  + 

+  + 
+  + 

++ 

++ 
++ 

H  — 

FEPTONIZTXG  GROUP. 

Culture  450       ...                .                .                     . 

++ 
++ 

4— 

H  — 

++ 

H  — 

Culture  45ti  

In  one  sample  of  milk  heated  at  82.2°  C.  (180°  F.)  for  30  minutes 
it  was  found  by  the  tube  method  of  group  differentiation  that  a  very 
large  percentage  of  the  acid  group  survived.  Sixty-four  milk  cul- 
tures of  the  resistant  acid  bacteria  were  examined  roughly  for  their 
thermal  death  point  by  inoculating  fresh  litmus  milk  tubes  with  one 
drop  of  the  milk  culture.  One  set  of  milk  tubes  was  then  heated  for 
30  minutes  at  87.8°  C.  (190°  F.)  in  a  water  bath  and  another  set  at 
93.3°  C.  (200°  F.)  for  the  same  length  of  time.  Of  the  64  tubes 
heated  at  87.8  C.  (190°  F.)  27  showed  growth,  but  all  were  destroyed 
at  93.3°  C.  (200°  F.).  These  heat-resistant  acid-forming  organisms 
are  of  great  importance  in  connection  with  the  pasteurization  of 
milk.  From  a  purely  scientific  standpoint  they  are  of  interest,  since 
in  general  the  thermal  death  point  of  vegetative  cells  has  been 
believed  to  be  below  60°  C.  (140°  F.).  From  the  numbers  of  these 
heat-resistant  organisms  it  is  evident  that  they  are  not  u-icommon  in 
milk. 

THE     POSSIBLE     USE     IN    PASTEURIZATION     OF     ACID  -  FORMING 
BACTERIA   OF   HIGH  THERMAL   DEATH   POINT. 

Since  certain  strains  of  typical  lactic-acid  bacteria  are  known  to  be 
able  to  withstand  pasteurizing  temperatures,  it  seems  possible  to 
make  use  of  their  resistance.  It  has  been  suggested  that  cultures  of 
lactic-acid  bacteria  be  added  to  milk  after  pasteurization  in  order  to 
insure  a  normal  souring.  A  process  of  that  nature  would  necessarily 
involve  a  danger  of  contamination  by  pathogenic  bacteria  from 
employees  at  the  dairy,  since  the  acid-forming  bacteria  are  to  be  added 
after  the  heating  process.  By  the  use  of  a  lactic-acid  organism  of 
high  thermal  death  point  it  would  be  possible  to  add  the  culture  to  the 


QUALITATIVE    STUDY    OF    BACTERIAL   FLORA. 


53 


raw  milk  and  then  pasteurize.  One  experiment  was  tried  to  show  this 
point.  As  may  be  seen  from  Table  20,  a  sample  of  certified  milk  was 
divided  into  two  parts  of  800  cubic  centimeters  each.  Both  portions 
were  heated  in  sterile  flasks  for  30  minutes  at  62.8°  C.  (145°  F.).  To 
one  flask  5  cubic  centimeters  of  a  high-temperature  resistant  lactic- 
acid  culture  was  added  before  pasteurization.  The  effect  of  the  addi- 
tion of  the  culture  can  be  seen  by  the  change  in  the  milk  when  held  at 
room  temperature.  After  four  days  the  milk  without  the  culture  was 
curdled  with  a  rennet  curd  and  was  partly  peptonized,  very  little  acid 
having  developed.  The  other  sample  to  which  the  heat  resistant  cul- 
ture was  added  was  curdled  on  the  fourth  day  and  showed  a  smooth 
firm  acid  curd  with  no  digestion.  By  the  addition  of  the  culture 
before  pasteurization  in  sealed  bottles  subsequent  infection  would  be 
impossible.  This  possibility  of  addition  of  high  temperature  resistant 
lactic-acid  bacteria  is  merely  suggested  for  those  who  advocate  the 
addition  of  cultures  after  pasteurization. 

TABLE  20. — Change  in  acid  development  in  pasteurized  milk  produced  by  the  addition  of 
high-temperature  resisting  lactic-acid  bacteria  previous  to  pasteurization. 

PER  CENT  LACTIC  ACID. 
[Samples  held  at  room  temperature,  21.1°-23.9°  C.  (70c-75°  F.).] 


Lactic  acid  after— 

0 

1 

2 

3 

4 

day. 

day. 

days. 

days. 

days. 

Perct. 

Perct. 

Perct. 

Perct. 

Perct. 

Certified  milk  pasteurized  at  (145°)  C2.8°  C.  for  30  minutes  
Some  of  same  milk  with  5  c.  c.  of  high-temperature  resisting  lactic-acid  cul- 

0.17 

0.17 

0.  10 

10.31 

ture  added  to  800  c.c.  of  milk;  milk  then  pasteurized  at  02.8°  C.  (145°  F.) 

for  30  minutes     

.17 

.17 

.  18 

2.50 

i  Curdled  and  peptonized. 


Curdled,  acid  curd. 


QUALITATIVE    STUDY  OF  THE   COMPLETE  BACTERIAL  FLORA  OF 
ONE    SAMPLE    OF   PASTEURIZED   MILK. 

Since  in  the  qualitative  study  of  the  bacteria  surviving  pasteuriza- 
tion a  few  typical  cultures  representing  each  group  were  selected  from 
each  sample,  it  was  thought  advisable  to  make  a  more  thorough  quali- 
tative study  of  the  bacteria  in  one  sample  of  pasteurized  milk. 

The  complete  bacterial  flora  of  one  sample  of  milk  pasteurized  for 
30  minutes  at  60°  C.  (140°  F.)  and  another  portion  of  the  same  milk 
pasteurized  at  65.6°  C.  (150°  F.)  were  studied  qualitatively. 

Litmus  lactose  gelatin  plates  were  made,  and  after  6  days'  incuba- 
tion at  18°  C.  (64.4°  F.)  each  colony  was  picked  off,  purified  by 
plating,  and  studied.  The  A  series  of  cultures  is  made  up  of  176  cul- 
tures picked  from  a  plate  from  milk  pasteurized  at  60°  C.  (140°  F.). 
The  B  series  is  composed  of  92  cultures  from  a  plate  from  milk  pas- 
teurized at  65.6°  (150°  F.).  Each  series  has  been  divided  into  five 


BACTERIA  WHICH   SURVIVE  PASTEURIZATION. 

IK 


QUALITATIVE   STUDY  OF  BACTERIAL  FLORA.  55 

divisions  according  to  the  differentiation  of  the  bacteria.  Table  21 
shows  the  A  series.  Division  1,  figure  27,  shows  the  division  of  the 
176  cultures  of  bacteria  which  survive  pasteurization  at  60°  C. 
(140°  F.)  into  three  groups — the  acid,  the  alkali,  and  the  peptonizing 
group. 

Per  cent. 

Acid  group 94.  88 

Alkali  group 1. 1 3 

Peptonizing  group •  3.  97 

In  division  2  the  acid  group  has  been  further  divided  as  shown  and 
the  percentages  given.  Each  division  shows  the  complete  percent- 
ages of  the  various  classes.  Division  5  shows  the  final  classification 
and  percentages.  Each  subdivision  of  division  5  has  been  given  a 
letter  which  makes  it  possible  by  the  use  of  the  following  key  to  deter- 
mine the  complete  reactions  of  the  bacteria  in  each  subdivision : 

KEY  TO   LETTERS   AND   SIGNS   USED   IN   FORMULA    SHOWING   CULTURAL 
REACTIONS    OF   THE    SUBDIVISIONS. 

The  formula  reads  in  the  order  of  the  numerals : 

1.  B— Bacilli. 
C— Cocci. 

2.  A — Gelatin  not  liquefied. 
X — Gelatin  liquefied. 

Xi-1-10  mm.  of  liquefaction. 
X2-over  10  mm.  of  liquefaction. 

3.  Gram (±) 

4.  Nitrate  reduced N  (±) 

5.  Dextrose  (sugar  broth) D  (± ) 

6.  Lactose  (sugar  broth) L  (±) 

7.  Saccharose  (sugar  broth) C  (±) 

8.  Galactose  (sugar  broth) G  (±) 

9.  Mannite M  (±) 

10.  Raffinose  (sugar  broth) R  (±) 

11.  Glycerin  broth Glyc.  (±) 

12.  Inulin  broth I  (±) 

13.  Starch  broth S  (±) 

14.  Pigment  production :  -  -P  (±) 

The  formulas  of  the  reactions  of  the  subdivisions  are  given  in 
Table  21. 


56 


BACTEBIA  WHICH    SURVIVE   PASTEURIZATION. 


QUALITATIVE    STUDY    OF    BACTERIAL   FLORA. 


57 


TABLE  21. — Cultural  reactions  of  organisms  in  subdivisions — cultures  of  A  series  from 
milk  pasteurized  for  30  minutes  at  60°  C.  (140°  F.). 


Subdivi- 
sion. 


Formula  of  reactions. 


CA+N-D+L+C±G+M±R-Glyc.-I-S±. 
CA-fN-D+L+C±G+M±R  +  Glyc.-I-S+. 
CA  +  N-D+L+C±G+M±R+Glyc.-I-S-. 
CA+N-D+L+C±G+M±R-Glyc.+I-S+. 
CA+N-D  +  L+C±G  +  M±R-Glyc.4l-S-. 
CA+N-D+L+C±G+M±R-Glyc.-I-S-P+. 
CA+N+D+L+C±G+M±R  +  Glyc.-I-S+. 
CA+N+D-f-L+C±G+M±R-Glye.-I-S-. 
CA  +  N4-  D+L+C±G+M±R+  Glyc.-I-S+. 


CA+N+D  +  L+C±G  f  MiR  +  Glye.-I-S-. 
CA+N+D+L+C±G  +  M±(other  reactions  (±)). 
CA-N-D+L+C±G+-M±R-Glyc.-I-S±. 
CA-N-D+L+C±G  +  M±(other  reactions  (±)). 


CA-N+D+L+C±G+M±R-Glvc.+I-S±. 
CA+N+D-L-C-G-M-R-Giyc.-I-S-P-Kred). 
CXs-N-D  +  L+C-f-G  +  M-R-Glye.-I-S-. 
CXs+N-D+L+C+G  +  M±R-Glyc.±I-S-I'±. 


The  formula  reads  as  follows,  as  shown  in  table: 

Subdivision  A : 
C.  Coccus. 

A.  Gelatin  not  liquefied. 
+  .  Gram+. 

N  — .  Nitrate  not  reduced. 
D-f-.  Acid  produced  in  dextrose  broth. 
L+.  Acid  produced  in  lactose  broth. 
C± .  May  or  may  not  produce  acid  in  saccharose  broth. 
G+.  Acid  produced  in  galactose  broth. 
M±.  May  or  may  not  produce  acid  in  mannite  broth. 
R  — .  No  acid  produced  in  raffinose  broth. 
Gly.  — .  No  acid  produced  in  glycerin  broth. 
I  —  .  No  acid  produced  in  inulin  broth. 
S±.  May  or  may  not  produce  acid  in  starch  broth. 

The  B  series  of  cultures  from  milk  pasteurized  at  65.6°  C.  (150°  F.) 
were  divided  in  a  similar  manner  as  shown  in  Fig.  28.  The  formulas 
of  the  reactions  of  the  subdivisions  are  given  in  Table  22. 

TABLE  22. — Cultural  reactions  of  organisms  in  subdivisions — cultures  of  B  series  from 
milk  pasteurized  for  SO  minutes  at  6.5.6°  C.  (150°  F.). 


Subdivi- 
sion. 

Formula  of  reactions. 

A 
It 
C 
D 
B 
F 
G 
H 
I 
J 
K 
L 
M 

CA+N-D+L+C±G+M±R-Glyc.-I-S±. 
CA+N-D+L+C±O4-M±R-^Glyc.-I-Sj-. 
CA+N-I-D+L+C±G  +  M±R+  Glyc.-I-S+. 
CA+N+D+L+C±G+M±R-t-Glyc.-I-S-. 
CA+N+D  +  L+C±G  +  M±R-Glyc.-I-S+. 
CA  +  N+D+L+C±G+M±R-Glyc.-I-S-. 
CA  +  N+D+L+C±G+M±R  +  Glyc.  +  I-S+. 
CA+N  +  D+L+C±G+M±R+Glyc.-I+S+. 
CA-N-D-t-L+C±G  +  M±R-Glyc.-I-S+. 
CA-N-D+L+C±G  +  M±R  +  Glvc.-l+S+. 
CA-N+r>+L+C±G  +  MiR  +  GIyc.-I-8+. 
CA-N+D+L+C-tG  +  M-R-Glye.-I-S+. 
CA-N-D-L-C-G-M-R-Glvc.-I-S-. 

58  BACTERIA    WHICH    SURVIVE   PASTEURIZATION. 

By  this  system  of  division  one  is  able  to  see  at  a  glance  the  general 
groups  of  bacteria  which  survived  pasteurization  in  one  sample  of 
milk  heated  at  60°  C.  (140°  F.)  and  65.6°  C.  (150°  F.).  Or  if  desired, 
the  complete  reactions  of  the  various  classes  of  bacteria  can  be  deter- 
mined. There  is  little  that  need  be  said  regarding  the  results,  for 
they  are  plainly  shown.  The  most  noticeable  features  were  the  high 
percentage  of  the  acid-forming  bacteria  and  the  low  percentage  of  the 
peptonizing  group.  It  may  be  seen  that  a  large  percentage  of  the 
acid  group  were  typical  lactic  acid  bacteria  as  based  on  the  formula 
of  subdivision  A. 

SUMMARY. 

1.  The  average  temperature  used  throughout  the  country  with 
the  "holder"  process  is  62.8°  C.  (145°  F.).    With  the  "flash"  process, 
71.1°C.  (160°  F.). 

Reports  from  219  milk  plants  show  that  75  used  the  "holder"  and 
144  the  "flash"  process.  The  range  in  temperatures  is  from  60°  C. 
(140°  F.)  to  82.2°  C.  (180°  F.).  Of  the  plants  using  the  "holder" 
process  only  1.3  per  cent  employ  temperatures  too  low  for  proper 
pasteurization.  Of  those  which  use  the  "flash"  process  approxi- 
mately 42  per  cent  use  temperatures  too  low  to  be  effective. 

2.  Percentage  bacterial  reduction  is  of  no  value  in  determining  the 
efficiency  of  the  process  of  pasteurization.    As  a  general  rule  when 
the  bacterial  content  of  raw  milk  is  high  there  will  be  a  high  per- 
centage reduction.    When  the  bacterial  content  is  low,  then  the  per- 
centage reduction  is  often  low.    The  percentage  bacterial  reduction 
may  be  99.9  per  cent  and  yet  the  pasteurized  milk  may  show  a  count 
of  100,000  per  cubic  centimeter.     When  the  pasteurized  milk  con- 
tains  only    10,000    bacteria   per   cubic   centimeter   the   percentage 
reduction  may  only  have  been  95  per  cent.    It  is  often  impossible  to 
obtain  a  99  per  cent  reduction  when  a  good  quality  of  milk  is  pas- 
teurized, therefore  regulations  which  require  a  99  per  cent  reduction 
of  bacteria  during  pasteurization  are  of  no  value. 

3.  A  heating  period  of  three  hours  causes  a  marked  increase  in  the 
reduction  of  bacteria  in  milk  over  one-half  hour's  heating,  when  a 
temperature  of  54.4°  C.  (130°  F.)  and  57.2°  C.  (135°  F.)  are  used. 
At  60°  C.  (140°  F.)  the  increased  reduction  is  much  less.    According 
to  the  results  of  one  experiment  in  this  investigation,  a  six  hours' 
period  of  heating  at  62.8°  C.  (145°  F.)  does  not  produce  any  more 
destruction  of  bacteria  than  does  one-half  hour's  heating. 

4.  Sudden  cooling  from  62.8°  C.  (145°  F.)  or  71.1°  C.  (160°  F.) 
within  15  seconds  to  from  1.7°-3.9°  C.  (35°-39°  F.j  does  not  cause 
any  destruction  of  bacteria.    The  long-prevailing  idea  that  sudden 
cooling  is  an  essential  part  of  the  process  of  pasteurization  is  errone- 
ous so  far  as  any  increased  destruction  of  bacteria  is  concerned.    Its 


QUALITATIVE  STUDY  OF  BACTERIAL  FLORA.  59 

value  lies  only  in  the  fact  that  milk  suddenly  cooled  is  not  allowed  to 
stand  at  temperatures  between  37.8°  C.  (100°  F.)  and  10°  C.  (50°  F.), 
where  rapid  bacterial  development  might  occur. 

5.  Four  distinct  groups  of  bacteria,  the  acid-forming,  the  inert, 
the  alkali-forming,  and  the  peptonizing,  survive  pasteurization,  as 
differentiated  by  their  reactions  in  litmus  milk  after  14  days'  incu- 
bation at  30°  C.  (86°  F.). 

The  percentage  of  the  acid  group  is  increased  by  pasteurization 
while  the  other  groups  are  decreased  in  their  percentage  of  the  total 
bacteria.  The  average  percentage  of  lactic  acid  bacteria  in  raw  milk 
which  survive  pasteurization  for  30  minutes  at  62.8°  C.  (145°  F.) 
ranges  from  1.27  per  cent  to  4.55  per  cent  in  the  various  grades  of 
milk. 

When  the  temperature  of  pasteurization  is  below  76.7°  C.  (170°  F.) 
the  bacterial  flora  of  milk  is  composed  for  the  most  part  of  organisms 
of  the  acid  group.  At  temperatures  above  76.7°  C.  (170°  F.)  the 
acid  group  is  largely  destroyed  and  the  majority  of  the  bacteria 
which  survive  are  of  the  peptonizing  type.  These  bacterial  group 
relations  are  clearly  shown  in  the  figures  in  the  conclusions. 

6.  When  different  grades  of  milk  are  pasteurized   at  62.8°  C. 
(145°  F.)  in  the  laboratory  and  held  at  room  temperature  the  bac- 
terial flora  may  undergo  three  distinct  changes.     First,  when  a  fair 
quality  of  milk  is  pasteurized  the  acid  group  may  develop  at  once  and 
overgrow  all  the  other  groups,  forming  acid  and  producing  a  normal 
curd.     Second,   when   a   poor   quality  of   milk  is   pasteurized   the 
peptonizing  group  may  grow  rapidly  at  first  along  with  the  acid 
group,  which  later  overgrows  them.     In  this  case  the  milk  will  first 
become  curdled  with  a  rennet  curd  due  to  the  peptonizing  bacteria, 
then  later  will  become  sour  from  the  development  of  the  lactic-acid 
group  of  organisms.     Third,  when  a  good  grade  of  milk  is  pasteur- 
ized the  peptonizing  bacteria  may  overgrow  the  acid  group  of  organ- 
isms so  that  the  milk  becomes  peptonized  without  the  develop- 
ment of  any  acid.    These  same  grades  of  milk,  treated  in  the  same 
manner  but  held  in  an  ice  chest  at  10°  C.  (50°  F.),  show  entirely 
different  changes  in  their  bacterial  contents.     The  growth  of  the 
peptonizing  group  is  restrained  so  that  they  are  of  little  importance. 
The  percentage  of  the  acid  group  remains  about  the  same  through  a 
long  period.     Occasionally  the  percentage  of  the  alkali  group  may 
increase  after  five  days,  but  eventually  the  acid  group  forms  the 
major  group.     These  results  were  obtained  from  laboratory  experi- 
ments and  only  indicate  possible  changes  in  the  bacterial  flora  of 
pasteurized  milk  when  held  at  different  temperatures.     They  show 
the  delicate  balance   between  the  bacterial   groups,  but  can  not 
be  applied  to  indicate  the  bacterial  changes  in  milk  pasteurized  and 
handled  under  commercial  conditions. 


60  BACTERIA   WHICH   SURVIVE   PASTEURIZATION. 

7.  A  qualitative  study  of  the  acid  group  shows  that  many  organ- 
isms giving  characteristics  of  the  typical  lactic-acid  bacteria  survive 
pasteurization  for  30  minutes  at  62.8°  C.  (145°  F.).     Other  lactic- 
acid  forming  bacteria  survive  which  may  form  pigment  or  vary  from 
the  typical  lactic  types  in  the  fermentative  reactions. 

The  inert  group  was  made  up  of  organisms  which  produce  no 
change  in  litmus  milk  during  14  days'  incubation,  and  therefore 
include  many  which  did  not  grow,  but  which  according  to  the  method 
of  differentiation  would  be  included  as  inert  forms.  The  only  truly 
inert  form  recognized  was  a  yellow  pigment-forming  organism.  The 
inert  group  is  probably  of  little  importance. 

The  alkali  group  which  survive  pasteurization  is  of  importance. 
Bacteria  of  this  group  can  not  be  differentiated  from  those  of  the 
inert  group  or  from  slow  acid-forming  organisms  by  plating,  but  are 
easily  determined  by  the  milk-tube  method.  They  grow  at  low 
temperatures  and  produce  a  strong  alkaline  reaction  in  milk.  In 
pure  cultures  enough  alkali  is  produced  to  dissolve  the  casein.  No 
acid  is  produced  in  fermentation  broths,  but  often  an  alkaline  reaction 
is  found. 

The  peptonizing  group  includes  those  organisms  which  peptonize 
casein.  A  number  of  the  organisms  isolated  peptonized  casein,  but 
did  not  liquefy  gelatin.  Various  types  of  this  group  are  able  to  sur- 
vive pasteurization  and  vary  widely  in  their  cultural  characteristics. 

The  few  gas-forming  bacteria  in  pasteurized  milk,  so  far  as  the 
results  of  this  investigation  indicate,  do  not  include  organisms  of  the 
colon-aerogenes  group.  One  gas-forming  organism  was  found  which 
produced  gas  in  milk  and  peptonized  casein.  Another  culture,  known 
as  Z,  seems  to  be  an  entirely  new  type,  which  is  characterized  by  its 
ability  to  produce  gas  continuously  in  milk  through  a  long  period, 
and  by  the  fact  that  while  gas  is  formed  in  milk  none  is  formed  in 
lactose  broth. 

Gas-forming  anaerobic  bacteria  are  often  found  in  milk  which  pro- 
duce gas  hi  lactose  bile  tubes  and  which  might  be  mistaken  in  a 
preliminary  test  for  the  colon  organism. 

8.  In  view  of  the  fact  that  gas-forming  organisms  survive  pas- 
teurization which  will  give  the  preliminary  test  for  the  colon  organ- 
ism by  the  lactose  bile  fermentation-tube  method,  it  seems  advisable 
to  suggest  that  boards  of  health  which  depend  on  the  presence  of  the 
colon-aerogenes  group  as  an  indication  of  reinfection  or  inefficient 
pasteurization  make  a  complete  determination  by  cultural  reactions 
of  organisms  suspected  as  being  colon  forms. 

9.  The  thermal  death  point  of  one  lactic  acid  organism  isolated 
during  this  investigation  was  79.4°  C.  (175°  F.)  when  a  broth  culture 
was  heated  in  Sternberg  bulbs  for  30  minutes.     The  thermal  death 
points  of  64  acid-forming  bacteria  from  one  sample  of  milk  which 


CONCLUSIONS.  61 

survived  pasteurization  for  30  minutes  at  82.2°  C.  (180°  F.)  deter- 
mined roughly  was  between  82.2°  C.  (180°  F.)  and  93.3°  C.  (200°  F.). 

10.  Only  a  small  percentage  of  spore-forming  bacteria  are  found 
in  milk  pasteurized  for  30  minutes  at  62.8°  C.  (145°  F.).     Of  225  cul- 
tures selected  at  random  which  were  studied,  only  3,  or  1.35  per  cent, 
formed  spores. 

11.  For  those  who  advocate  the  inoculation  of  pasteurized  milk 
after  heating,  with  a  culture  of  lactic  acid  bacteria  to  produce  a 
normal  souring,  it  is  suggested  that  a  culture  of  a  high-temperature 
resisting  lactic-acid  organism  may  be  added  before  pasteurization. 
By  such  a  method  there  would  be  no  danger  of  infection  as  might 
result  accidentally  by  inoculation  after  pasteurization. 

12.  In  view  of  the  results  of  this  investigation  it  seems  that  the 
control  of  pasteurization  should  be  maintained  by  bacterial  limits 
for  the  milk  to  be  pasteurized,  together  with  supervision  which  will 
insure  the  use  of  the  proper  pasteurizing  temperature  and  guard 
against  reinfection.     A  bacterial  standard  then  need  not  be  set  for 
pasteurized  milk. 

CONCLUSIONS. 

The  results  in  this  paper  are  based  upon  laboratory  experiments 
performed  under  conditions  which  made  reinfection  impossible. 
The  bacteria  which  survived  pasteurization,  therefore,  represent  those 
bacteria  which  could  not  be  destroyed  at  the  temperature  of  pasteuriza- 
tion. A  temperature  of  62.8°  C.  (145°  F.)  for  30  minutes  was  used 
in  most  of  the  experiments,  since  that  is  the  most  universally  used 
temperature  with  the  " holder"  process  in  this  country.  Higher 
temperatures,  however,  were  also  used  in  order  to  show  the  changes 
in  the  bacterial  groups  which  survived.  From  the  results  of  the 
work  it  has  been  possible  to  show  graphically  in  figure  29  the  hypo- 
thetical relations  of  the  bacterial  groups  in  raw  milk  and  milk  pas- 
teurized by  the  "holder"  process  at  various  temperatures  under 
laboratory  conditions.  The  bacterial  flora  of  the  various  kinds  of 
milk  is  represented  by  columns  of  equal  length  divided  into  sections, 
which  represent  in  a  general  way  the  relative  size  of  the  bacterial 
groups. 

From  the  figure  it  may  be  seen  that  raw  milk  contains  four  prin- 
cipal groups  of  bacteria — the  acid,  the  inert,  alkali,  and  peptonizing 
groups.  The  acid  group  is  divided  into  two  groups — the  acid-coagu- 
lating, which  coagulates  milk  in  less  than  14  days,  and  the  acid 
group,  which  merely  produces  acid  and  does  not  coagulate  milk 
inside  of  14  days.  In  raw  milk  the  inert  group  is  the  largest.  In 
milk  pasteurized  at  62.8°  C.  (145°  F.)  the  great  increase  in  the  pro- 
portion of  the  acid-coagulating  and  acid  groups  is  plainly  shown. 
The  percentage  of  the  alkali  and  peptonizing  groups  are  reduced. 
At  71.1°  C.  (160°  F.)  the  total  acid  group  is  still  the  largest,  but 


62 


BACTERIA   WHICH    SURVIVE   PASTEURIZATION. 


the  acid  coagulating  group  is  made  up  of  bacteria  which  coagulate 
very  slowly.  At  this  temperature  the  alkali  group  is  greatly  reduced 
and  the  peptonizing  reduced  to  the  minimum.  At  76.7°  C.  (170°  F.) 
the  total  acid  group  remains  about  the  same,  but  the  organisms  pro- 
duce acid  and  coagulate  very  slowly.  The  alkali  group  is  practically 


CCMGVL4T//VG 
<S#OVf> 


&P7VWZWG 
GffOf/ff 


30 


I 


i 


767°C.       82.2°C.     87.8°C.      _ 

(/8o°/r)     (/9o°/y    (200°^) 


11 


vjr 

0 

c^ 

fu 

i 

k 

I 

I 

i 

FIG.  29. — The  hypothetical  relation  of  the  bacterial  groups  to  raw  and  pasteurized  milk. 

destroyed,  although  occasionally  a  sample  may  show  a  fairly  high 
percentage.  The  most  important  change  is  in  the  peptonizing  group. 
At  this  temperature  the  percentage  of  the  peptonizing  group  to  the 
total  bacteria  begins  to  increase.  The  increase  when  milk  is  pas- 
teurized at  82.2°  C.  (180°  F.)  is  even  more  striking.  At  this  tem- 
perature over  75  per  cent  of  the  bacteria  which  survive  are  pep- 


CONCLUSIONS. 


63 


Ionizers.  None  of  the  acid-coagulating  group  are  found  and  only 
a  small  percentage  of  the  acid  group.  Occasionally  a  few  of  the 
alkali  group  may  be  found.  At  87.8°  C.  (190°  F.)  and  93.3°  C. 


/oo 


FIG.  30.— The  hypothetical  relation  of  the  bacterial  groups  in  raw  and  pasteurized  milk. 

(200°  F.)  the  bacterial  groups  which  survive  are  about  the  same 
in  their  relative  sizes  as  at  82.2°  C.  (180°  F.).  The  hypothetical  rela- 
tions of  the  bacterial  groups  are  further  shown  in  figure  30.  The  rela- 


64  BACTERIA   WHICH    SURVIVE   PASTEURIZATION. 

tion  of  the  bacterial  groups  is  also  shown,  which  brings  out  more 
distinctly,  perhaps,  the  changes  in  the  bacterial  groups.  Here 
the  reversal  of  the  percentages  of  the  total  acid  group  and  the  pep- 
tonizing  group  in  milk  pasteurized  above  76.7°  C.  (170°  F.)  for  30 
minutes  is  plainly  shown.  It  is  very  evident  that  when  the  bac- 
terial flora  of  pasteurized  milk  is  under  discussion  the  temperature 
of  the  process  is  of  fundamental  importance.  From  these  figures 
the  bacterial  groups  in  various  kinds  of  milk  may  be  seen  at  a 
glance,  and  they  summarize  in  a  very  general  way  the  most 
important  results  of  this  investigation.  It  must  be  remembered, 
however,  that  the  relations  of  the  bacterial  groups  only  represent 
average  conditions,  and  the  bacterial  flora  of  every  sample  of  milk 
must  not  be  expected  to  conform  exactly  to  these  averages.  Varia- 
tions in  methods  and  conditions  in  the  production  of  milk  beyond 
the  control  of  the  investigator  may  widely  influence  the  bacterial 
group  relations  of  an  individual  sample. 


APPENDIX. 


65 


APPENDIX. 


TABLE  I.  —  Complete  characteristics  of  the  different   varieties  of  acid-forming   bacteria 
which  survive  pasteurization. 


Acid  production  in— 


3 

s 

a  i  s 


>  Yellow. 


66 


BACTERIA   WHICH   SURVIVE   PASTEURIZATION. 


TABLE  II. — Complete  characteristics  of  the  different  varieties  of  alkali-forming  bacteria 
which  survive  pasteurization. 


WWWOOOO  Morphology. 

1 

O 

Gelatin  liquefied 
or  nonliquefied. 

Nitrate  reduced. 

"3 

E 

Acid  production  in— 

Alkaline  reaction 
in  litmus  milk. 

Dextrose. 

a 

Saccharose. 

Galactose. 

Mannite. 

d 

! 

3 

Rafflnose. 

Inulin. 

Starch. 

Salicin. 

+ 

4 
4 

- 

4- 

1  4 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

! 

- 

4 

'  4 

- 

-  • 

- 

- 

- 

- 

- 

- 

- 

- 

Yellow. 


TABLE  III. — Complete  characteristics  of  the  different  varieties  of  the  peptonizing  bacteria 
which  survive  pasteurization. 


Acid  production  in— 


Yellow. 


Brown. 


Dark  cream. 


1  Spores. 


O 


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